Process for producing dipeptides

ABSTRACT

The present invention provides a protein which catalyzes the synthesis of a dipeptide different from L-Ala-L-Ala, a process for producing the protein which catalyzes the synthesis of a dipeptide, a process for producing a dipeptide using the protein which catalyzes the synthesis of a dipeptide, and a process for producing the dipeptide using a culture of a microorganism producing the protein which catalyzes the synthesis of a dipeptide or the like as an enzyme source.

The present application claims benefit of Japanese application nos.376054/2002 and 420887/2003, filed 26 Dec. 2002 and 18 Dec. 2003,respectively, the entire contents of each of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a protein which catalyzes the synthesisof a dipeptide, a process for producing the protein which catalyzes thesynthesis of a dipeptide, a process for producing a dipeptide using theprotein which catalyzes the synthesis of a dipeptide, a microorganism ora transformant producing the protein which catalyzes the synthesis of adipeptide, and a process for producing the dipeptide using suchmicroorganism or transformant.

Chemical synthesis methods (liquid phase method and solid phase method),enzymatic synthesis methods and biological synthesis methods utilizingrecombinant DNA techniques are available for large-scale peptidesynthesis. Currently, the enzymatic synthesis methods and biologicalsynthesis methods are employed for the synthesis of long-chain peptideslonger than 50 residues, and the chemical synthesis methods andenzymatic synthesis methods are mainly employed for the synthesis ofdipeptides.

In the synthesis of dipeptides by the chemical synthesis methods,however, operations such as introduction and removal of protectivegroups for functional groups are necessary, and racemates are alsoformed. The chemical synthesis methods are thus considered to bedisadvantageous in respect of cost and efficiency. They are unfavorablealso from the viewpoint of environmental hygiene because of the use oflarge amounts of organic solvents and the like.

As to the synthesis of dipeptides by the enzymatic methods, thefollowing methods are known: a method utilizing reverse reaction ofprotease (J. Biol. Chem., 119, 707-720 (1937)); methods utilizingthermostable aminoacyl t-RNA synthetase (Japanese Published UnexaminedPatent Application No. 146539/83, Japanese Published Unexamined PatentApplication No. 209991/83, Japanese Published Unexamined PatentApplication No. 209992/83,and Japanese Published Unexamined PatentApplication No. 106298/84); and methods utilizing non-ribosome peptidesynthetase (hereinafter referred to as NRPS) (Chem. Biol., 7, 373-384(2000), FEBS Lett., 498, 42-45 (2001), U.S. Pat. No. 5,795,738 and U.S.Pat. No. 5,652,116).

However, the method utilizing reverse reaction of protease requiresintroduction and removal of protective groups for functional groups ofamino acids used as substrates, which causes difficulties in raising theefficiency of peptide-forming reaction and in preventingpeptide-degradating reaction. The methods utilizing thermostableaminoacyl t-RNA synthetase have the defects that the expression of theenzyme and the prevention of side reactions forming by-products otherthan the desired products are difficult. The methods utilizing NRPS areinefficient in that the expression of the enzyme by recombinant DNAtechniques is difficult because of its large enzyme molecule size andthat the supply of coenzyme 4′-phosphopantetheine is necessary.

On the other hand, there exist a group of peptide synthetases that haveenzyme molecular weight lower than that of NRPS and do not requirecoenzyme 4′-phosphopantetheine; for example, γ-glutamylcysteinesynthetase, glutathione synthetase, D-alanine-D-alanine (D-Ala-D-Ala)ligase, and poly-γ-glutamate synthetase. Most of these enzymes utilizeD-amino acids as substrates or catalyze peptide bond formation at theγ-carboxyl group. Because of such properties, they can not be used forthe synthesis of dipeptides by peptide bond formation at the α-carboxylgroup of L-amino acid.

The only known example of an enzyme capable of dipeptide synthesis bythe activity to form a peptide bond at the α-carboxyl group of L-aminoacid is bacilysin (dipeptide antibiotic derived from a microorganismbelonging to the genus Bacillus ) synthetase. Bacilysin synthetase isknown to have the activity to synthesize bacilysin[L-alanyl-L-anticapsin (L-Ala-L-anticapsin)] and L-alanyl-L-alanine(L-Ala-L-Ala), but there is no information about its activity tosynthesize other peptides (J. Ind. Microbiol., 2, 201-208 (1987) andEnzyme. Microbial. Technol., 29, 400-406 (2001)).

As for the bacilysin biosynthetase genes in Bacillus subtilis 168 whoseentire genome information has been clarified (Nature, 390, 249-256(1997)), it is known that the productivity of bacilysin is increased byamplification of bacilysin operons containing ORFs ywfA-F (WO00/03009pamphlet). However, it is not known whether an ORF encoding a proteinhaving the activity to ligate two or more amino acids by peptide bond iscontained in these ORFs, and it contained, which ORF encodes theprotein.

An object of the present invention is to provide a protein whichcatalyzes the synthesis of a dipeptide which is different fromL-Ala-L-Ala and for which no enzymatic synthesis method using peptidesynthetase or the like has so far been proposed, and a protein for thesynthesis of the dipeptide; DNA encoding the protein having thedipeptide-synthesizing activity; a recombinant DNA comprising the DNA; atransformant carrying the recombinant DNA; a process for producing theprotein having the dipeptide-synthesizing activity; an enzymatic methodfor synthesizing the dipeptide using the protein having thedipeptide-synthesizing activity or the protein for the dipeptidesynthesis; and a process for producing the dipeptide using, as an enzymesource, a culture of a microorganism having the ability to produce theprotein having the dipeptide-synthesizing activity or the protein forthe dipeptide synthesis, or the like.

An object of the present invention is to provide a protein whichcatalyzes the synthesis of a dipeptide which is different fromL-Ala-L-Ala and for which no enzymatic synthesis method using peptidesynthetase or the like has so far been proposed, and a protein for thesynthesis of the dipeptide; DNA encoding the protein having thedipeptide-synthesizing activity; a recombinant DNA comprising the DNA; atransformant carrying the recombinant DNA; a process for producing theprotein having the dipeptide-synthesizing activity; an enzymatic methodfor synthesizing the dipeptide using the protein having thedipeptide-synthesizing activity or the protein for the dipeptidesynthesis; and a process for producing the dipeptide using, as an enzymesource, a culture of a microorganism having the ability to produce theprotein having the dipeptide-synthesizing activity or the protein forthe dipeptide synthesis, or the like.

SUMMARY OF THE INVENTION

The present invention relates to the following (1) to (20).

(1) A protein selected from the group consisting of [1] to [4] below,provided that a protein consisting of the amino acid sequence shown inSEQ ID NO: 1 is excluded:

-   -   [1] a protein comprising the amino acid sequence shown in any of        SEQ ID NOS: 2 to 8;    -   [2] a protein comprising an amino acid sequence wherein one or        more amino acid residues are deleted, substituted or added in        the amino acid sequence shown in any of SEQ ID NOS: 1 to 8 and        catalyzing the synthesis of a dipeptide represented by formula        (I):        R¹—R²  (I)    -   (wherein R¹ and R², which may be the same or different, each        represent an amino acid, provided that both R¹ and R² cannot        represent L-alanine at the same time);    -   [3] a protein comprising an amino acid sequence which shows 65%        or more similarity to the amino acid sequence shown in any of        SEQ ID NOS: 1 to 8 and catalyzing the synthesis of dipeptide        represented by formula (I); and    -   [4] a protein comprising an amino acid sequence which shows 80%        or more similarity to the amino acid sequence shown in SEQ ID        NO: 17 and catalyzing the synthesis of a dipeptide represented        by formula (I).

(2) A nucleic acid sequence selected from the group consisting of [1] to[5] below, provided that the nucleotide sequence shown in SEQ ID NO: 9is excluded, and the RNA equivalent thereof, are:

-   -   [1] a nucleic acid sequence encoding the protein according to        the above (1);    -   [2] a nucleic acid sequence comprising the nucleotide sequence        shown in any of SEQ ID NOS: 10 to 16 and 36;    -   [3] a nucleic acid sequence which hybridizes with a nucleic acid        sequence comprising the complement of a nucleotide sequence        shown in any of SEQ ID NOS: 9 to 16 and 36 under stringent        conditions and which nucleic acid sequence encodes a protein        which catalyzes the synthesis of a dipeptide represented by        formula (I):        R¹—R²  (I)        (wherein R¹ and R², which may be the same or different, each        represent an amino acid, provided that both R¹ and R² cannot        represent L-alanine at the same time);    -   [4] a nucleic acid sequence comprising a nucleotide sequence        which shows 80% or more similarity to the nucleotide sequence        shown in SEQ ID NO: 18 and encoding a protein which catalyzes        the synthesis of a dipeptide represented by formula (I); and    -   [5] a nucleic acid sequence which hybridizes with a nucleic acid        sequence of any one of SEQ ID NOs: 9 to 16 and 36 under        stringent conditions and is useful as a probe or primer for        identifying, detecting, amplifying, etc. a nucleic acid sequence        which catalyzes the synthesis of a dipeptide represented by        formula (I).

(3) A recombinant nucleic acid sequence comprising the nucleic acidsequence according to the above (2).

(4) A transformant carrying the recombinant nucleic acid sequenceaccording to the above (3).

(5) The transformant according to the above (4), wherein thetransformant is a transformant obtained by using a microorganism as ahost.

(6) The transformant according to the above (5), wherein themicroorganism is a microorganism belonging to the genus Escherichia.

(7) A process for producing the protein according to the above (1),which comprises culturing the transformant according to any of the above(4) to (6) in a medium, allowing the protein according to the above (1)to form and accumulate in the culture medium, and recovering the proteinfrom the culture medium.

(8) A process for producing the protein according to the above (1),which comprises culturing a microorganism having the ability to producethe protein according to the above (1) in a medium, allowing the proteinto form and accumulate in the culture medium, and recovering the proteinfrom the culture medium.

(9) The process according to the above (8), wherein the microorganism isa microorganism belonging to the genus Bacillus.

(10) The process according to the above (9), wherein the microorganismbelonging to the genus Bacillus is a microorganism having the ability toproduce bacilysin.

(11) The process according to the above (10), wherein the microorganismhaving the ability to produce bacilysin is a microorganism belonging toa species selected from the group consisting of Bacillus subtilis,Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis,Bacillus megaterium and Bacillus pumilus.

(12) A process for producing a dipeptide represented by formula (I):R¹—R²  (I)(wherein R¹ and R², which may be the same or different, each representan amino acid, provided that both R¹ and R² cannot represent L-alanineat the same time), which comprises:

-   -   allowing the protein according to the above (1) or a protein        comprising the amino acid sequence shown in SEQ ID NO:1, at        least two amino acids which may be the same or different, and        ATP to be present in an aqueous medium;    -   allowing the dipeptide to form and accumulate in the medium; and    -   recovering the dipeptide from the medium.

(13) A process for producing a dipeptide represented by formula (I):R¹—R²  (I)(wherein R¹ and R², which may be the same or different, each representan amino acid, provided that both R¹ and R² cannot represent L-alanineat the same time), which comprises:

-   -   allowing an enzyme source and at least two amino acids which may        be the same or different to be present in an aqueous medium,        said enzyme source being a culture or a treated matter of the        culture selected from the group consisting of the following [1]        to [3]:    -   [1] a culture of the transformant according to any of the        above (4) to (6) or a treated matter of the culture;    -   [2] a culture of a microorganism having the ability to produce        the protein according to the above (1) or a treated matter of        the culture; and    -   [3] a culture of a microorganism having the ability to produce        the protein comprising the amino acid sequence shown in SEQ ID        NO:1 or a treated matter of the culture;    -   allowing the dipeptide to form and accumulate in the medium; and    -   recovering the dipeptide from the medium.

(14) The process according to the above (13), wherein the microorganismhaving the ability to produce the protein according to the above (1) isa microorganism belonging to the genus Bacillus .

(15) The process according to the above (14), wherein the microorganismbelonging to the genus Bacillus is a microorganism of the genus Bacillushaving the ability to produce bacilysin.

(16) The process according to the above (15), wherein the microorganismof the genus Bacillus having the ability to produce bacilysin is amicroorganism belonging to a species selected from the group consistingof Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus coagulans,Bacillus licheniformis, Bacillus megaterium and Bacillus pumilus.

(17) The process according to the above (13), wherein the microorganismhaving the ability to produce the protein comprising the amino acidsequence shown in SEQ ID NO:1 is a microorganism transformed with arecombinant nucleic acid sequence comprising a nucleic acid sequencecomprising the nucleotide sequence shown in SEQ ID NO: 9 or amicroorganism belonging to Bacillus subtilis.

(18) The process according to the above (17), wherein the microorganismtransformed with a recombinant nucleic acid sequence comprising anucleic acid sequence comprising the nucleotide sequence shown in SEQ IDNO: 9 is a microorganism belonging to the genus Escherichia.

(19) The process according to any of the above (13) to (18), wherein thetreated matter of the culture is a concentrated culture, a driedculture, cells obtained by centrifuging the culture, or a productobtained by subjecting the cells to drying, freeze-drying, treatmentwith a surfactant, ultrasonication, mechanical friction, treatment witha solvent, enzymatic treatment, protein fractionation or immobilization,or an enzyme preparation obtained by extracting the cells.

(20) The process according to any of the above (12) to (19), wherein thedipeptide is a dipeptide represented by formula (II):R³—R⁴   (II)(wherein R³ and R⁴, which may be the same or different, each representan amino acid selected from the group consisting of L-alanine,L-glutamine, L-glutamic acid, glycine, L-valine, L-leucine,L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine,L-serine, L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine,L-arginine, L-histidine, L-aspartic acid, L-α-aminobutyric acid,β-alanine, L-azaserine, L-theanine, L-4-hydroxyproline,L-3-hydroxyproline, L-ornithine, L-citrulline andL-6-diazo-5-oxo-norleucine, provided that both R³ and R⁴ cannotrepresent L-alanine at the same time).

In accordance with the present invention, a protein which catalyzes thesynthesis of a dipeptide which is different from L-Ala-L-Ala and forwhich no enzymatic synthesis method has so far been proposed can beproduced. Dipeptides other than L-Ala-L-Ala can be produced by using theprotein, or a transformant or a microorganism having the ability toproduce the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the steps for constructing plasmid pPE43.

FIG. 2 shows the steps for constructing plasmid pQE60ywfE.

EXPLANATION OF SYMBOLS

ywfE: ywfE gene derived from Bacillus subtilis 168

Ptrp: Tryptophan promoter gene

PT5: T5 promoter gene

DETAILED DESCRIPTION OF THE INVENTION

The proteins of the present invention include:

[1] a protein comprising the amino acid sequence shown in any of SEQ IDNOS: 2 to 8;

[2] a protein comprising an amino acid sequence wherein one or moreamino acid residues are deleted, substituted or added in the amino acidsequence shown in any of SEQ ID NOS: 1 to 8 and catalyzing the synthesisof a dipeptide represented by formula (I)R¹—R²  (I)(wherein R¹ and R², which may be the same or different, each representan amino acid, provided that both R¹ and R² cannot represent L-alanineat the same time)

[3] a protein comprising an amino acid sequence which shoes 65% or moresimilarity to the amino acid sequence shown in any of SEQ ID NOS: 1 to 8and catalyzing the synthesis of a dipeptide represented by formula (I);and

[4] a protein comprising an amino acid sequence which shows 80% or moresimilarity to the amino acid sequence shown in SEQ ID NO: 17 andcatalyzing the synthesis of a dipeptide represented by formula (I),provided that a protein consisting of the amino acid sequence shown inSEQ ID NO: 1 is excluded

The proteins of the present invention are useful of the production of adipeptide of formula (I), for example, as may be provided by the proteinof SEQ ID NO: 1.

The use of the protein of SEQ ID NO: 1 in this manner forms a part ofthe present invention along with the further products and methodsdescribed herein.

Hereinafter, the above proteins of the present invention and proteinswhich catalyzes the synthesis of a dipeptide represented by formula (I)may be collectively referred to as the proteins of the presentinvention.

The above protein consisting of an amino acid sequence wherein one ormore amino acid residues are deleted, substituted or added andcatalyzing the synthesis of a dipeptide represented by formula (I) canbe obtained, for example, by introducing a site-directed mutation intoDNA encoding a protein consisting of the amino acid sequence shown inany of SEQ ID NOS: 1 to 8 by site-directed mutagenesis described inMolecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989) (hereinafter referred to as MolecularCloning, Second Edition); Current Protocols in Molecular Biology, JohnWiley & Sons (1987-1997) (hereinafter referred to as Current Protocolsin Molecular Biology); Nucleic Acids Research, 10, 6487 (1982); Proc.Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985); NucleicAcids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488(1985), etc.

The number of amino acid residues which are deleted, substituted oradded is not specifically limited, but is within the range wheredeletion, substitution or addition is possible by known methods such asthe above site-directed mutagenesis. The suitable number is 1 to dozens,preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5.

The expression “one or more amino acid residues are deleted, substitutedor added in the amino acid sequence shown in any of SEQ ID NOS: 1 to 8”means that the amino acid sequence may contain deletion, substitution oraddition of a single or plural amino acid residues at an arbitraryposition of the amino acid sequence shown in SEQ ID NO: 1 to 8.

Amino acid residues that may be substituted are, for example, thosewhich are not conserved in all of the amino acid sequences shown in SEQID NOS: 1 to 8 when the sequences are compared using known alignmentsoftware. An example of known alignment software is alignment analysissoftware contained in gene analysis software Genetyx (SoftwareDevelopment Co., Ltd.). As analysis parameters for the analysissoftware, default values can be used.

Deletion or addition of amino acid residues may be contained, forexample, in the N-terminal region or the C-terminal region of the aminoacid sequence shown in any of SEQ ID NOS: 1 to 8.

Deletion, substitution and addition may be simultaneously contained inone sequence, and amino acids to be substituted or added may be eithernatural or not. Examples of the natural amino acids are L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine and L-cysteine.

The following are examples of the amino acids capable of mutualsubstitution. The amino acids in the same group can be mutuallysubstituted.

-   -   Group A: leucine, isoleucine, norleucine, valine, norvaline,        alanine, 2-aminobutanoic acid, methionine, O-methylserine,        t-butylglycine, t-butylalanine, cyclohexylalanine    -   Group B: aspartic acid, glutamic acid, isoaspartic acid,        isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid    -   Group C: asparagine, glutamine    -   Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,        2,3-diaminopropionic acid    -   Group E: proline, 3-hydroxyproline, 4-hydroxyproline    -   Group F: serine, threonine, homoserine    -   Group G: phenylalanine, tyrosine

In order that the protein of the present invention may have the activityto synthesize a dipeptide represented by formula (I), it is desirablethat the similarity of its amino acid sequence to the amino acidsequence shown in any of SEQ ID NOS: 1 to 8, preferably SEQ ID NO: 1, is65% or more, preferably 75% or more, more preferably 85% or more,further preferably 90% or more, particularly preferably 95% or more, andmost preferably 98% or more.

The similarity among amino acid sequences and nucleotide sequences canbe determined by using algorithm BLAST by Karlin and Altschul [Proc.Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183,63 (1990)]. On the basis of the algorithm BLAST, programs such as BLASTNand BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When anucleotide sequence is analyzed by BLASTN on the basis of BLAST, theparameters, for instance, are as follows: score=100 and wordlength=12.When an amino acid sequence is analyzed by BLASTX on the basis of BLAST,the parameters, for instance, are as follows: score=50 and wordlength=3.When BLAST and Gapped BLAST programs are used, default parameters ofeach program are used. The specific techniques for these analyses areknown (see, the internet site: ncbi.nlm.nih.gov.).

A protein consisting of an amino acid sequence which shows 65% or more,preferably 75% or more, more preferably 85% or more, further preferably90% or more, particularly preferably 95% or more, most preferably 98% ormore similarity to the amino acid sequence shown in any of SEQ ID NOS: 1to 8, preferably SEQ ID NO: 1, and catalying the synthesis of adipeptide represented by formula (I) is also included in the proteins ofthe present invention (provided that a protein consisting of the aminoacid sequence shown in SEQ ID NO: 1 is excluded). The similarity amongamino acid sequences can be determined by using BLAST or FASTA asdescribed above.

The amino acid sequence shown in SEQ ID NO: 17 is a region conservedamong the proteins having the amino acid sequences shown in SEQ ID NOS:1 to 7 and is also a region corresponding to the consensus sequence ofproteins having Ala-Ala ligase activity derived from variousmicroorganisms.

A protein comprising an amino acid sequence which shows 80% or more,preferably 90% or more, further preferably 95% or more similarity to theamino acid. sequence shown in SEQ ID NO: 17 and catalyzing the synthesisof a dipeptide represented by formula (I) is also included in theproteins of the present invention (provided that a protein consisting ofthe amino acid sequence shown in SEQ ID NO: 1 is excluded).

In order that the protein comprising an amino acid sequence which shows80% or more, preferably 90% or more, further preferably 95% or moresimilarity to the amino acid sequence shown in SEQ ID NO: 17 may havethe activity to synthesize a dipeptide represented by formula (I), it isdesirable that the similarity of its amino acid sequence to the aminoacid sequence shown in any of SEQ ID NOS: 1 to 8 is at least 80% ormore, usually 90% or more, and particularly 95% or more.

The similarity among amino acid sequences can be determined by usingBLAST or FASTA as described above.

It is possible to confirm that the protein of the present invention is aprotein which catalyzes the synthesis of a dipeptide represented by theabove formula (I), for example, in the following manner. That is, atransformant expressing the protein of the present invention is preparedby recombinant DNA techniques, the protein of the present invention isproduced using the transformant, and then the protein of the presentinvention, at least two amino acids which may be the same or different(provided that L-alanine is used in combination with another L-aminoacid) and ATP are allowed to be present in an aqueous medium, followedby HPLC analysis or the like to know whether a dipeptide represented bythe above formula (I) is formed and accumulated in the aqueous medium.

The nucleic acid sequences of the present invention include:

-   -   [5] a nucleic acid sequence encoding the protein of the present        invention according to any of the above [1] to [4];    -   [6] a nucleic acid sequence comprising the nucleotide sequence        shown in any of SEQ ID NOS: 10 to 16 and 36;    -   [7] a nucleic acid sequence which hybridizes with a nucleic acid        sequence comprising the complement of a nucleotide sequence        shown in any of SEQ ID NOS: 9 to 16 and 36 under stringent        conditions and which encodes a protein which catalyzes the        synthesis of a dipeptide represented by formula (I), provided        that DNA consisting of the nucleotide sequence shown in SEQ ID        NO: 9 is excluded, preferably provided that DNA encoding the        protein consisting of the amino acid sequence shown in SEQ ID        NO: 1 is excluded; and    -   [8] a nucleic acid sequence comprising a nucleotide sequence        which shows 80% or more similarity to the nucleotide sequence        shown in SEQ ID NO: 18 and encoding a protein which catalyzes        the synthesis of a dipeptide represented by formula (I),        provided that DNA consisting of the nucleotide sequence shown in        SEQ ID NO: 9 is excluded, preferably provided that DNA encoding        the protein consisting of an amino acid sequence shown in SEQ ID        NO: 1 is excluded.

The nucleic acid sequences that can be used in the process for producinga dipeptide represented by formula (I) of the present invention includethe nucleic acid sequences according to the above [5] to [8] and anucleic acid sequence comprising the nucleotide sequence shown in SEQ IDNO: 9.

The above nucleic acid sequence capable of hybridization under stringentconditions refers, for example, to a nucleic acid sequences which areobtained by colony hybridization, plaque hybridization, Southern blothybridization, or the like using a part or the whole of a nucleic acidsequence comprising the nucleotide sequence shown in any of SEQ ID NOS:9 to 16 and 36 as a probe. A specific example of such a nucleic acidsequence is a nucleic acid sequence which can be identified byperforming hybridization at 65° C. in the presence of 0.7 to 1.0 mol/lsodium chloride using a filter with colony- or plaque-derived DNAimmobilized thereon, and then washing the filter at 65° C. with a 0.1 to2-fold conc. SSC solution (1-fold conc. SSC solution: 150 mmol/l sodiumchloride and 15 mmol/l sodium citrate). Hybridization can be carried outaccording to the methods described in Molecular Cloning, Second Edition;Current Protocols in Molecular Biology; DNA Cloning 1: Core Techniques,A Practical Approach, Second Edition, Oxford University (1995), etc.Specifically, the hybridizable nucleic acid sequences includes DNAhaving at least 75% or more similarity, preferably 85% or moresimilarity, further preferably 90% or more similarity, particularlypreferably 95% or more similarity to the nucleotide sequence shown inany of SEQ ID NOS: 9 to 16 and 36 as calculated by use of BLAST or FASTAdescribed above based on the above parameters.

It is possible to confirm that the nucleic acid sequence whichhybridizes with a nucleic acid sequence comprising the nucleotidesequence shown in any of SEQ ID NOS: 9 to 16 and 36 under stringentconditions is a nucleic acid sequence encoding a protein which catalyzesthe synthesis of a dipeptide represented by formula (I), for example, byproducing a protein encoded by the DNA by recombinant DNA techniques andmeasuring the activity of the protein as described above.

(i) Preparation of a DNA of the Present Invention and DNA Used in aProcess for Producing the Protein or Dipeptide of the Present Invention

A DNA of the present invention and a DNA used in the process forproducing the protein or dipeptide of the present invention(hereinafter, also referred to as the production process of the presentinvention) can be obtained, for example, by Southern hybridization of achromosomal DNA library from a microorganism belonging to the genusBacillus using a probe designed based on the nucleotide sequence shownin any of SEQ ID NOS: 9 to 16 and 36, or by PCR [PCR Protocols, AcademicPress (1990)] using primer DNAs designed based on the nucleotidesequence shown in any of SEQ ID NOS: 9 to 16 and 36 and, as a template,the chromosomal DNA of a microorganism belonging to the genus Bacillus.

A DNA of the present invention and a DNA used in the production processof the present invention can also be obtained by conducting a searchthrough various gene sequence databases for a sequence showing 75% ormore similarity, preferably 85% or more similarity, more preferably 90%or more similarity, further preferably 95% or more similarity,particularly preferably 98% or more similarity to the nucleotidesequence of DNA encoding the amino acid sequence shown in any of SEQ IDNOS: 1 to 8 and 17, and obtaining the desired DNA, based on thenucleotide sequence obtained by the search, from a chromosomal DNA orcDNA library of an organism having the nucleotide sequence according tothe above-described method.

The obtained DNA, as such or after cleavage with appropriate restrictionenzymes, is inserted into a vector by a conventional method, and theobtained recombinant DNA is introduced into a host cell. Then, thenucleotide sequence of the DNA can be determined by a conventionalsequencing method such as the dideoxy method [Proc. Natl. Acad. Sci.,USA, 74, 5463 (1977)] or by using a nucleotide sequencer such as 373ADNA Sequencer (Perkin-Elmer Corp.).

In cases where the obtained DNA is found to be a partial DNA by theanalysis of nucleotide sequence, the full length DNA can be obtained bySouthern hybridization of a chromosomal DNA library using the partialDNA as a probe.

It is also possible to prepare the desired DNA by chemical synthesisusing a DNA synthesizer (e.g., Model 8905, PerSeptive Biosystems) basedon the determined nucleotide sequence of the DNA.

Examples of the DNAs that can be obtained by the above-described methodare DNAs having the nucleotide sequences shown in SEQ ID NOS: 9 to 16and 36.

Examples of the vectors for inserting the DNA of the present inventionor the DNA used in the production process of the present inventioninclude pBluescriptII KS(+) (Stratagene), pDIRECT [Nucleic Acids Res.,18, 6069 (1990)], pCR-Script Amp SK(+) (Stratagene), pT7 Blue (Novagen,Inc.), pCR II (Invitrogen Corp.) and pCR-TRAP (Genhunter Corp.).

As the host cell, microorganisms belonging to the genus Escherichia,etc. can be used. Examples of the microorganisms belonging to the genusEscherichia include Escherichia coli XL1-Blue, Escherichia coliXL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichiacoli ATCC 12435, Escherichia coli W1485, Escherichia coli JM109,Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110,Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522and Escherichia coli ME8415.

Introduction of the recombinant DNA can be carried out by any of themethods for introducing DNA into the above host cells, for example, themethod using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)],the protoplast method (Japanese Published Unexamined Patent ApplicationNo. 248394/88) and electroporation [Nucleic Acids Res., 16, 6127(1988)].

An example of the microorganism carrying the DNA used in the productionprocess of the present invention obtained by the above method isEscherichia coli NM522/pPE43, which is a microorganism transformed witha recombinant DNA comprising DNA having the sequence shown in SEQ ID NO:1.

(ii) Process for Producing a Protein of the Present Invention

A protein of the present invention can be produced by expressing a DNAof the present invention or a DNA used in the production process of thepresent invention obtained by the methods described in the above (i) inhost cells using the methods described in Molecular cloning, SecondEdition, Current Protocols in Molecular Biology, etc., for example, inthe following manner.

On the basis of a DNA of the present invention or a DNA used in theproduction process of the present invention, a nucleic acid sequence,such as a DNA, fragment of an appropriate length comprising a regionencoding a protein of the present invention is prepared according toneed. The productivity of the protein can be enhanced by replacing anucleotide in the nucleotide sequence of the region encoding the proteinso as to make a codon most suitable for the expression in a host cell.Codon optimized nucleic acid sequences therefore will be recognized asbeing a part of the presently disclosed invention.

The DNA fragment may be inserted downstream of a promoter in anappropriate expression vector to prepare a recombinant DNA.

A transformant producing a protein of the present invention can beobtained by introducing the recombinant DNA into a host cell suited forthe expression vector.

As a host cell, any bacterial cells, yeast cells, animal cells, insectcells, plant cells, etc. that are capable of expressing the desirednucleic acid squence can be used.

The expression vectors that can be employed are those capable ofautonomous replication or integration into the chromosome in the abovehost cells and comprising, for example, a promoter at a positionappropriate for the transcription of a DNA of the present invention or aDNA used in the production process of the present invention.

When a procaryote such as a bacterium is used as the host cell, it ispreferred that a recombinant DNA comprising a DNA of the presentinvention or a DNA used in the production process of the presentinvention is a recombinant DNA which is capable of autonomousreplication in the procaryote and which comprises, for example, apromoter, a ribosome binding sequence, a DNA of the present invention ora DNA used in the production process of the present invention, and atranscription termination sequence. The recombinant DNA may furthercomprise a gene or nucleic acid sequence regulating the promoter.

Examples of suitable expression vectors are pBTrp2, pBTac1 and pBTac2(products of Boehringer Mannheim GmbH), pHelix1 (Roche DiagnosticsCorp.), pKK233-2 (Amersham Pharmacia Biotech), pSE280 (InvitrogenCorp.), pGEMEX-1 (Promega Corp.), pQE-8 (Qiagen, Inc.), pET-3 (Novagen,Inc.), pKYP10 (Japanese Published Unexamined Patent Application No.110600/83), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric.Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 92,4306 (1985)], pBluescript II SK(+), pBluescript II KS(−) (Stratagene),pTrs30 [prepared from Escherichia coliJM109/pTrS30 (FERM BP-5407)],pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)],pPAC31 (WO98/12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara ShuzoCo., Ltd.), pUC118 (Takara Shuzo Co., Ltd.) and pPA1 (Japanese PublishedUnexamined Patent Application No. 233798/88).

As the promoter, any promoters capable of functioning in host cells suchas Escherichia coli can be used. For example, promoters derived fromEscherichia coli or phage, such as trp promoter (P_(trp)), lac promoter(P_(lac)), P_(L) promoter, P_(R) promoter and P_(SE) promoter, SPO1promoter, SPO2 promoter and penP promoter can be used. Artificiallydesigned and modified promoters such as a promoter in which two P_(trp)sare combined in tandem, tac promoter, lacT7 promoter and letI promoter,etc. can also be used.

Also useful are xylA promoter for the expression in microorganismsbelonging to the genus Bacillus [Appl. Microbiol. Biotechnol., 35,594-599 (1991)] and P54-6 promoter for the expression in microorganismsbelonging to the genus Corynebacterium [Appl. Microbiol. Biotechnol.,53, 674-679 (2000)].

It is preferred to use a plasmid in which the distance between theShine-Dalgarno sequence (ribosome binding sequence) and the initiationcodon is adjusted to an appropriate length (e.g., 6 to 18 nucleotides).

In the recombinant DNA wherein the DNA of the present invention or theDNA used in the production process of the present invention is ligatedto an expression vector, the transcription termination sequence is notessential, but it is preferred to place the transcription terminationsequence immediately downstream of the structural gene.

An example of such recombinant DNA is pPE43.

Examples of suitable procaryotes include microorganisms belonging to thegenera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium,Microbacterium, Pseudomonas, Agrobacterium, Alicyclobacillus, Anabaena,Anacystis, Arthrobacter, Azotobacter, Chromatium, Erwinia,Methylobacterium, Phormidium, Rhodobacter, Rhodopseudomonas,Rhodospirillum, Scenedesmus, Streptomyces, Synechoccus and Zymomonas.Specific examples are Escherichia coli XL1-Blue, Escherichia coliXL2-Blue, Escherichia coli DH1, Escherichia coli DH5α, Escherichia coliMC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichiacoli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichiacoli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichiacoli NM522, Bacillus subtilis ATCC 33712, Bacillus megaterium, Bacillussp. FERM BP-6030, Bacillus amyloliguefaciens, Bacillus coagulans,Bacillus licheniformis, Bacillus pumilus, Brevibacterium ammoniagenes,Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticumATCC 14066, Brevibacterium flavum ATCC 14067, Brevibacteriumlactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13032,Corynebacterium glutamicum ATCC 14297, Corynebacterium acetoacidophilumATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Serratia ficaria,Serratia fonticola, Serratia liquefaciens, Serratia marcescens,Pseudomonas sp D-0110, Agrobacterium radiobacter, Agrobacteriumrhizogenes, Agrobacterium rubi, Anabaena cylindrica, Anabaena doliolum,Anabaena flos-aquae, Arthrobacter aurescens, Arthrobacter citreus,Arthrobacter globformis, Arthrobacter hydrocarboglutamicus, Arthrobactermysorens, Arthrobacter nicotianae, Arthrobacter paraffineus,Arthrobacter protophormiae, Arthrobacter roseoparaffinus, Arthrobactersulfureus, Arthrobacter ureafaciens, Chromatium buderi, Chromatiumtepidum, Chromatium vinosum, Chromatium warmingii, Chromatiumfluviatile, Erwinia uredovora, Erwinia carotovora, Erwinia ananas,Erwinia herbicola, Erwinia punctata, Erwinia terreus, Methylobacteriumrhodesianum, Methylobacterium extorquens, Phormidium sp. ATCC 29409,Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodopseudomonasblastica, Rhodopseudomonas marina, Rhodopseudomonas palustris,Rhodospirillum rubrum, Rhodospirillum salexigens, Rhodospirillumsalinarum, Streptomyces ambofaciens, Streptomyces aureofaciens,Streptomyces aureus, Streptomyces fungicidicus, Streptomycesgriseochromogenes, Streptomyces griseus, Streptomyces lividans,Streptomyces olivogriseus, Streptomyces rameus, Streptomycestanashiensis, Streptomyces vinaceus and Zymomonas mobilis.

Introduction of the recombinant DNA can be carried out by any of themethods for introducing DNA into the above host cells, for example, themethod using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)],the protoplast method (Japanese Published Unexamined Patent ApplicationNo. 248394/88) and electroporation [Nucleic Acids Res., 16, 6127(1988)].

When a yeast strain is used as the host cell, YEp13 (ATCC 37115), YEp24(ATCC 37051), YCp50 (ATCC 37419), pHS19, pHS15, etc. can be used as theexpression vector.

As the promoter, any promoters capable of functioning in yeast strainscan be used. Suitable promoters include PHO5 promoter, PGK promoter, GAPpromoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shockpolypeptide promoter, MFα1 promoter and CUP 1 promoter.

Examples of suitable host cells are yeast strains belonging to thegenera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon,Schwanniomyces, Pichia and Candida, specifically, Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris andCandida utilis.

Introduction of the recombinant DNA can be carried out by any of themethods for introducing DNA into yeast, for example, electroporation[Methods Enzymol., 194, 182 (1990)], the spheroplast method [Proc. Natl.Acad. Sci. USA, 81, 4889 (1984)] and the lithium acetate method [J.Bacteriol., 153, 163 (1983)].

When an animal cell is used as the host cell, pcDNAI, pcDM8(commercially available from Funakoshi Co., Ltd.), pAGE107 (JapanesePublished Unexamined Patent Application No. 22979/91), pAS3-3 (JapanesePublished Unexamined Patent Application No. 227075/90), pCDM8 [Nature,329, 840 (1987)], pcDNAI/Amp (Invitrogen Corp.), pREP4 (InvitrogenCorp.), pAGE103 [J. Biochem., 101, 1307 (1987)], pAGE210, pAMo, pAMoA,etc. can be used as the expression vector.

As the promoter, any promoters capable of functioning in animal cellscan be used. Suitable promoters include the promoter of IE (immediateearly) gene of cytomegalovirus (CMV), SV40 early promoter,metallothionein promoter, the promoter of a retrovirus, heat shockpromoter, SRα promoter, etc. The enhancer of IE gene of human CMV may beused in combination with the promoter.

Examples of suitable host cells are mouse myeloma cells, rat myelomacells, mouse hybridomas, human-derived Namalwa cells and Namalwa KJM-1cells, human embryonic kidney cells, human leukemia cells, African greenmonkey kidney cells, Chinese hamster-derived CHO cells, and HBT5637(Japanese Published Unexamined Patent Application No. 299/88).

The mouse myeloma cells include SP2/0 and NSO; the rat myeloma cellsinclude YB2/0; the human embryonic kidney cells include HEX293 (ATCCCRL-1573); the human leukemia cells include BALL-1; and the Africangreen monkey kidney cells include COS-1 and COS-7.

Introduction of the recombinant DNA can be carried out by any of themethods for introducing DNA into animal cells, for example,electroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphatemethod (Japanese Published Unexamined Patent Application No. 227075/90),lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and themethod described in Virology, 52, 456 (1973).

When an insect cell is used as the host cell, the protein can beproduced by using the methods described in Baculovirus Expressionvectors, A Laboratory Manual, W. H. Freeman and Company, New York(1992); Current Protocols in Molecular Biology; Molecular Biology, ALaboratory Manual; Bio/Technology, 6, 47 (1988), etc.

That is, the recombinant gene transfer vector and a baculovirus arecotransfected into insect cells to obtain a recombinant virus in theculture supernatant of the insect cells, and then insect cells areinfected with the recombinant virus, whereby the protein can beproduced.

The gene transfer vectors useful in this method include pVL1392, pVL1393and pBlueBacIII (products of Invitrogen Corp.).

An example of the baculovirus is Autographa californica nuclearpolyhedrosis virus, which is a virus infecting insects belonging to thefamily Barathra.

Examples of the insect cells are ovarian cells of Spodoptera frugiperda,ovarian cells of Trichoplusia ni, and cultured cells derived fromsilkworm ovary.

The ovarian cells of Spodoptera frugiperda include Sf9 and Sf21(Baculovirus Expression Vectors, A Laboratory Manual); the ovarian cellsof Trichoplusia ni include High 5 and BTI-TN-5B1-4 (Invitrogen Corp.);and the cultured cells derived from silkworm ovary include Bombyx moriN4.

Cotransfection of the above recombinant gene transfer vector and theabove baculovirus into insect cells for the preparation of therecombinant virus can be carried out by the calcium phosphate method(Japanese Published Unexamined Patent Application No. 227075/90),lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], etc.

When a plant cell is used as the host cell, Ti plasmid, tobacco mosaicvirus vector, etc. can be used as the expression vector.

As the promoter, any promoters capable of functioning in plant cells canbe used. Suitable promoters include 35S promoter of cauliflower mosaicvirus (CaMV), rice actin 1 promoter, etc.

Examples of suitable host cells are cells of plants such as tobacco,potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.

Introduction of the recombinant vector can be carried out by any of themethods for introducing DNA into plant cells, for example, the methodusing Agrobacterium (Japanese Published Unexamined Patent ApplicationNos. 140885/84 and 70080/85, WO94/00977), electroporation (JapanesePublished Unexamined Patent Application No. 251887/85) and the methodusing particle gun (gene gun) (Japanese Patent Nos. 2606856 and2517813).

When the DNA is expressed in yeast, an animal cell, an insect cell or aplant cell, a glycosylated protein can be obtained.

The protein of the present invention can be produced by culturing thetransformant obtained as above in a medium, allowing a protein of thepresent invention to form and accumulate in the culture, and recoveringthe protein from the culture.

The host of the above transformant for producing the protein of thepresent invention may be any bacterium, yeast, animal cell, insect cell,plant cell or the like, but is preferably a bacterium, more preferably amicroorganism belonging to the genus Escherichia, and further preferablya microorganism belonging to Escherichia coli.

Culturing of the above transformant in a medium can be carried out byconventional methods for culturing the host.

For the culturing of the transformant obtained by using a procaryotesuch as Escherichia coli or a eucaryote such as yeast as the host, anyof natural media and synthetic media can be used insofar as it is amedium suitable for efficient culturing of the transformant whichcontains carbon sources, nitrogen sources, inorganic salts, etc. whichcan be assimilated by the host used.

As the carbon sources, any carbon sources that can be assimilated by thehost can be used. Examples of suitable carbon sources includecarbohydrates such as glucose, fructose, sucrose, molasses containingthem, starch and starch hydrolyzate; organic acids such as acetic acidand propionic acid; and alcohols such as ethanol and propanol.

As the nitrogen sources, ammonia, ammonium salts of organic or inorganicacids such as ammonium chloride, ammonium sulfate, ammonium acetate andammonium phosphate, and other nitrogen-containing compounds can be usedas well as peptone, meat extract, yeast extract, corn steep liquor,casein hydrolyzate, soybean cake, soybean cake hydrolyzate, and variousfermented microbial cells and digested products thereof.

Examples of the inorganic salts include potassium dihydrogenphosphate,dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate andcalcium carbonate.

Culturing is usually carried out under aerobic conditions, for example,by shaking culture or submerged spinner culture under aeration. Theculturing temperature is preferably 15 to 40° C., and the culturingperiod is usually 5 hours to 7 days. The pH is maintained at 3.0 to 9.0during the culturing. The pH adjustment is carried out by using anorganic or inorganic acid, an alkali solution, urea, calcium carbonate,ammonia, etc.

If necessary, antibiotics such as ampicillin and tetracycline may beadded to the medium during the culturing.

When a microorganism transformed with an expression vector comprising aninducible promoter is cultured, an inducer may be added to the medium,if necessary. For example, in the case of a microorganism transformedwith an expression vector comprising lac promoter,isopropyl-β-D-thiogalactopyranoside or the like may be added to themedium; and in the case of a microorganism transformed with anexpression vector comprising trp promoter, indoleacrylic acid or thelike may be added.

For the culturing of the transformant obtained by using an animal cellas the host cell, generally employed media such as RPMI1640 medium [J.Am. Med. Assoc., 199, 519 (1967)], Eagle's MEM [Science, 122, 501(1952)], DMEM [Virology, 8, 396 (1959)] and 199 medium [Proc. Soc. Biol.Med., 73, 1 (1950)], media prepared by adding fetal calf serum or thelike to these media, etc. can be used as the medium.

Culturing is usually carried out at pH 6 to 8 at 25 to 40° C. for 1 to 7days in the presence of 5% CO₂.

If necessary, antibiotics such as kanamycin, penicillin and streptomycinmay be added to the medium during the culturing.

For the culturing of the transformant obtained by using an insect cellas the host cell, generally employed media such as TNM-FH medium(PharMingen, Inc.), Sf-900 II SFM medium (Life Technologies, Inc.),ExCell 400 and ExCell 405 (JRH Biosciences, Inc.) and Grace's InsectMedium [Nature, 195, 788 (1962)] can be used as the medium.

Culturing is usually carried out at pH 6 to 7 at 25 to 30° C. for 1 to 5days.

If necessary, antibiotics such as gentamicin may be added to the mediumduring the culturing.

The transformant obtained by using a plant cell as the host cell may becultured in the form of cells as such or after differentiation intoplant cells or plant organs. For the culturing of such transformant,generally employed media such as Murashige-Skoog (MS) medium and Whitemedium, media prepared by adding phytohormones such as auxin andcytokinin to these media, etc. can be used as the medium.

Culturing is usually carried out at pH 5 to 9 at 20 to 40° C. for 3 to60 days.

If necessary, antibiotics such as kanamycin and hygromycin may be addedto the medium during the culturing.

As described above, a protein of the present invention can be producedby culturing, according to a conventional culturing method, thetransformant derived from a microorganism, an insect cell, an animalcell or a plant cell and carrying a recombinant DNA prepared by ligatinga DNA of the present invention or a DNA used in the production processof the present invention to an expression vector, allowing the proteinto form and accumulate, and recovering the protein from the culture.

A protein of the present invention may be produced by intracellularproduction by host cells, extracellular secretion by host cells orproduction on outer membranes by host cells, and depending on the methodselected, the structure of the protein produced is suitably modified.

When a protein of the present invention is produced in host cells or onouter membranes of host cells, it is possible to force the protein to besecreted outside the host cells by applying the method of Paulson, etal. [J. Biol. Chem., 264, 17619 (1989)], the method of Lowe, et al.[Proc. Natl. Acad. Sci. USA, 86, 8227 (1989); Genes Develop., 4, 1288(1990)], or the methods described in Japanese Published UnexaminedPatent Application No. 336963/93, WO94/23021, etc.

That is, extracellular secretion of a protein of the present inventionby host cells can be caused by producing it in the form of a protein inwhich a signal peptide is added upstream of a protein containing theactive site of the protein of a present invention by the use ofrecombinant DNA techniques.

It is also possible to increase the protein production by utilizing agene amplification system using a dihydrofolate reductase gene or thelike according to the method described in Japanese Published unexaminedPatent Application No. 227075/90.

Further, a protein of the present invention can be produced using ananimal having an introduced gene (non-human transgenic animal) or aplant having an introduced gene (transgenic plant) constructed byredifferentiation of animal or plant cells carrying the introduced geneor nucleic acid sequence.

When a transformant producing a protein of the present invention is ananimal or plant, the protein can be produced by raising or culturing theanimal or plant in a usual manner, allowing the protein to form andaccumulate therein, and recovering the protein from the animal or plant.

Production of a protein of the present invention using an animal can becarried out, for example, by producing the protein in an animalconstructed by introducing the gene according to known methods [Am. J.Clin. Nutr., 63, 639S (1996); Am. J. Clin. Nutr., 63, 627S (1996);Bio/Technology, 9, 830 (1991)].

In the case of an animal, a protein of the present invention can beproduced, for example, by raising a non-human transgenic animal carryingan introduced DNA of the present invention or DNA used in the productionprocess of the present invention, allowing the protein to form andaccumulate in the animal, and recovering the protein from the animal.The places where the protein is formed and accumulated include milk(Japanese Published Unexamined Patent Application No. 309192/88), egg,etc. of the animal. As the promoter in this process, any promoterscapable of functioning in an animal can be used. Preferred promotersinclude mammary gland cell-specific promoters such as α casein promoter,β casein promoter, β lactoglobulin promoter and whey acidic proteinpromoter.

Production of the protein of the present invention using a plant can becarried out, for example, by culturing a transgenic plant carrying theintroduced DNA encoding the protein of the present invention accordingto known methods [Soshiki Baiyo (Tissue Culture), 20 (1994); SoshikiBaiyo, 21 (1995); Trends Biotechnol., 15, 45 (1997)], allowing theprotein to form and accumulate in the plant, and recovering the proteinfrom the plant. Tissue specific promoters may also be used in plantproduction by means known to one of ordinary skill.

Isolation and purification of a protein of the present inventionproduced using the transformant producing a protein of the presentinvention can be carried out by conventional methods for isolating andpurifying enzymes.

For example, when a protein of the present invention is produced in asoluble form in cells, the cells may be recovered by centrifugationafter the completion of culturing and suspended in an aqueous buffer,followed by disruption using a sonicator, French press, Manton Gaulinhomogenizer, Dynomill or the like to obtain a cell-free extract.

A purified protein preparation can be obtained by centrifuging thecell-free extract to obtain the supernatant and then subjecting thesupernatant to ordinary means for isolating and purifying enzymes, e.g.,extraction with a solvent, salting-out with ammonium sulfate, etc.,desalting, precipitation with an organic solvent, anion exchangechromatography using resins such as diethylaminoethyl (DEAE)-Sepharoseand DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchangechromatography using resins such as S-Sepharose FF (Pharmacia),hydrophobic chromatography using resins such as butyl Sepharose andphenyl Sepharose, gel filtration using a molecular sieve, affinitychromatography, chromatofocusing, and electrophoresis such asisoelectric focusing, alone or in combination.

When the protein is produced as an inclusion body in cells, the cellsare similarly recovered and disrupted, followed by centrifugation toobtain a precipitate fraction. After the protein is recovered from theprecipitate fraction by an ordinary method, the inclusion body of theprotein is solubilized with a protein-denaturing agent.

The solubilized protein solution is diluted with or dialyzed against asolution containing no protein-denaturing agent or a solution containingthe protein-denaturing agent at such a low concentration thatdenaturation of protein is not caused, whereby the protein is renaturedto have normal higher-order structure. Then, a purified proteinpreparation can be obtained by the same isolation and purification stepsas described above.

When a protein of the present invention or its derivative such as aglycosylated form is extracellularly secreted, the protein or itsderivative such as a glycosylated form can be recovered in the culturesupernatant.

That is, the culture is treated by the same means as above, e.g.,centrifugation, to obtain a soluble fraction. A purified proteinpreparation can be obtained from the soluble fraction by using the sameisolation and purification methods as described above.

Examples of the proteins obtained in the above manner are proteinsrespectively consisting of the amino acid sequences shown in SEQ ID NOS:1 to 8.

It is also possible to produce a protein of the present invention as afusion protein with another protein and to purify it by affinitychromatography using a substance having affinity for the fused protein.For example, according to the method of Lowe, et al. [Proc. Natl. Acad.Sci. USA, 86, 8227 (1989); Genes Develop., 4, 1288 (1990)] and themethods described in Japanese Published Unexamined Patent ApplicationNo. 336963/93 and WO94/23021, a protein of the present invention can beproduced as a fusion protein with protein A and can be purified byaffinity chromatography using immunoglobulin G.

Further, it is possible to produce a protein of the present invention asa fusion protein with a Flag peptide and to purify it by affinitychromatography using anti-Flag antibody [Proc. Natl. Acad. Sci. USA, 86,8227 (1989); Genes Develop., 4, 1288 (1990)]. The protein can also bepurified by affinity chromatography using an antibody against saidprotein.

A protein of the present invention can also be produced by chemicalsynthetic methods such as the Fmoc method (thefluorenylmethyloxycarbonyl method) and the tBoc method (thet-butyloxycarbonyl method) based on the amino acid sequence informationon the protein obtained above. Further, a protein of the presentinvention can be chemically synthesized by using peptide synthesizersfrom Advanced ChemTech, Perkin-Elmer, Pharmacia, Protein TechnologyInstrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, etc.

(iii) Process for Producing a Dipeptide of the Present Invention

(1) Enzymatic Process

An example of the enzymatic process for producing a dipeptide is aprocess which comprises: allowing a protein of the present invention, atleast two amino acids which may be the same or different, and ATP to bepresent in an aqueous medium; allowing a dipeptide represented byformula (I) to form and accumulate in the medium; and recovering thedipeptide from the medium.

At least two amino acids, preferably one or two kinds of amino acidsused as substrates in the above process are selected from the groupconsisting of amino acids, preferably L-amino acids, glycine (Gly) andβ-alanine (β-Ala), and can be used in any combination except for the useof L-alanine as one kind of amino acid. Examples of L-amino acids areL-alanine (L-Ala), L-glutamine (L-Gln), L-glutamic acid (L-Glu),L-valine (L-Val), L-leucine (L-Leu), L-isoleucine (L-Ile), L-proline(L-Pro), L-phenylalanine (L-Phe), L-tryptophan (L-Trp), L-methionine(L-Met), L-serine (L-Ser), L-threonine (L-Thr), L-cysteine (L-Cys),L-asparagine (L-Asn), L-tyrosine (L-Tyr), L-lysine (L-Lys), L-arginine(L-Arg), L-histidine (L-His), L-aspartic acid (L-Asp), L-α-aminobutyricacid (L-α-AB), L-azaserine, L-theanine, L-4-hydroxyproline (L-4-HYP),L-3-hydroxyproline (L-3-HYP), L-ornithine (L-Orn), L-citrulline (L-Cit)and L-6-diazo-5-oxo-norleucine.

The amino acids which are more preferably used in the above processinclude the following: a combination of one kind of amino acid selectedfrom the group consisting of L-Ala, Gly, L-Met, L-Ser, L-Thr and β-Ala,and one kind of amino acid selected from the group consisting of L-Ala,L-Gln, L-Glu, Gly, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp, L-Met,L-Ser, L-Thr, L-Cys, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp, L-α-AB,O-Ala, L-azaserine, L-theanine, L-4-HYP, L-3-HYP, L-Orn, L-Cit andL-6-diazo-5-oxo-norleucine (excluding a combination of L-Ala and L-Ala);a combination of L-Gln and L-Phe; and a combination of L-α-AB and L-Gln,L-Arg or L-α-AB. Further preferred amino acids are: a combination ofL-Ala and one kind of amino acid selected from the group consisting ofL-Gln, Gly, L-Val, L-Leu, L-Ile, L-Phe, L-Trp, L-Met, L-Ser, L-Thr,L-Cys, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-α-AB, L-azaserine, L-Cit andL-theanine; a combination of Gly and one kind of amino acid selectedfrom the group consisting of L-Gln, Gly, L-Phe, L-Trp, L-Met, L-Ser,L-Thr, L-Cys, L-Tyr, L-Lys, L-Arg, L-α-AB and L-Cit; a combination ofL-Met and one kind of amino acid selected from the group consisting ofL-Phe, L-Met, L-Ser, L-Thr, L-Cys, L-Tyr, L-Lys and L-His; a combinationof L-Ser and one kind of amino acid selected from the group consistingof L-Gln, L-Phe, L-Ser, L-Thr, L-Tyr, L-His and L-α-AB; a combination ofL-Thr and one kind of amino acid selected from the group consisting ofL-Gln, L-Phe, L-Leu, L-Thr and L-α-AB; a combination of L-Gln and L-Phe;a combination of β-Ala and one kind of amino acid selected from thegroup consisting of L-Phe, L-Met, L-His and L-Cit; and a combination ofL-a-AB and L-Gln, L-Arg or L-α-AB.

In the above process, a protein of the present invention is added in anamount of 0.01 to 100 mg, preferably 0.1 to 10 mg per mg of amino acidused as a substrate.

In the above process, the amino acid used as a substrate is added to theaqueous medium at the start or in the course of reaction to give aconcentration of 0.1 to 500 g/l, preferably 0.2 to 200 g/l.

In the above process, ATP used as an energy source is used at a finalconcentration of 0.5 mmol to 10 mol/l.

The aqueous medium used in the above process may comprise any componentsand may have any composition so far as the dipeptide-forming reaction isnot inhibited. Suitable aqueous media include water and buffers such asphosphate buffer, carbonate buffer, acetate buffer, borate buffer,citrate buffer and Tris buffer. The aqueous medium may comprise alcoholssuch as methanol and ethanol, esters such as ethyl acetate, ketones suchas acetone, and amides such as acetamide.

The dipeptide-forming reaction is carried out in the aqueous medium atpH 5 to 11, preferably pH 6 to 10, at 20 to 50° C., preferably 25 to 45°C., for 2 to 150 hours, preferably 6 to 120 hours.

The dipeptides produced by the above process include the dipeptidesrepresented by formula (I). Preferred dipeptides are those representedby formula (I) wherein R¹ and R², which may be the same or different,each represent an amino acid selected from the group consisting ofL-Ala, L-Gln, L-Glu, Gly, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp,L-Met, L-Ser, L-Thr, L-Cys, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp,L-α-AB, β-Ala, L-azaserine, L-theanine, L-4-HYP, L-3-HYP, L-Orn, L-Citand L-6-diazo-5-oxo-norleucine (excluding that wherein both R¹ and R²are L-Ala at the same time). More preferred are dipeptides wherein R¹ isL-Ala, Gly, L-Met, L-Ser, L-Thr or β-Ala, and R² is L-Gln, L-Glu, Gly,L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp, L-Met, L-Ser, L-Thr, L-Cys,L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp, L-α-AB, β-Ala, L-azaserine,L-theanine, L-4-HYP, L-3-HYP, L-Orn, L-Cit orL-6-diazo-5-oxo-norleucine. Further preferred dipeptides are: dipeptideswherein R¹ is L-Ala and R² is L-Gln, Gly, L-Val, L-Leu, L-Ile, L-Phe,L-Trp, L-Met, L-Ser, L-Thr, L-Cys, L-Asn, L-Tyr, L-Lys, L-Arg, L-His,L-α-AB, L-azaserine, L-theanine or L-Cit; dipeptides wherein R¹ is Glyand R² is L-Gln, Gly, L-Phe, L-Trp, L-Met, L-Ser, L-Thr, L-Cys, L-Tyr,L-Lys, L-Arg, L-α-AB or L-Cit; dipeptides wherein R¹ is L-Met and R² isL-Phe, L-Met, L-Cys, L-Tyr, L-Lys or L-His; dipeptides wherein R¹ isL-Ser and R² is L-Gln, Gly, L-Phe, L-Met, L-Ser, L-Thr, L-Tyr, L-His orL-α-AB; dipeptides wherein R¹ is L-Thr and R² is L-Gln, L-Leu, L-Phe,L-Met, L-Ser, L-Thr or L-α-AB; dipeptides wherein R¹ is L-Gln and R² isL-Phe or L-α-AB; a dipeptide wherein R¹ is L-Phe and R² is L-Gln; adipeptide wherein R¹ is L-Trp and R² is Gly; dipeptides wherein R¹ isL-Cys and R² is L-Ala, L-Gln, Gly or L-Met; dipeptides wherein R¹ isL-Lys and R² is L-Ala, Gly or L-Met; dipeptides wherein R¹ is β-Ala andR² is L-Phe, L-Met or L-His; a dipeptide wherein R¹ is L-Arg and R² isL-α-AB; a dipeptide wherein R¹ is L-His and R² is L-Met; and dipeptideswherein R¹ is L-α-AB and R² is L-Ala, L-Gln, Gly, L-Ser, L-Thr, L-Arg orL-α-AB.

(2) Process using a Culture of a Transformant or Microorganism or aTreated Matter of the Culture as an Enzyme Source

An example of the process for producing a dipeptide using a culture of atransformant or microorganism or a treated matter of the culture as anenzyme source is a process which comprises: allowing an enzyme sourceand at least two amino acids which may be same or different to bepresent in an aqueous medium, said enzyme source being a culture of atransformant having the ability to produce a protein of the presentinvention or a microorganism having the ability to produce a protein ofthe present invention, or a treated matter of the culture; allowing adipeptide represented by formula (I) to form and accumulate in themedium; and recovering the dipeptide from the medium.

Transformants useful in the above process include the transformantsproducing a protein of the present invention that can be produced by themethod of the above (ii). As the hosts of the transformants, bacteria,yeast, animal cells, insect cells, plant cells, etc. can be used.Preferred hosts are bacteria, among which microorganisms belonging tothe genera Escherichia, Bacillus and Corynebacterium are more preferred.

Preferred microorganisms belonging to the genus Escherichia includethose belonging to Escherichia coli; preferred microorganisms belongingto the genus Bacillus include those belonging to Bacillus subtilis andBacillus megaterium; and preferred microorganisms belonging to the genusCorynebacterium include those belonging to Corynebacterium glutamicumand Corynebacterium ammoniagenes.

The microorganism used in the above process may be any microorganismhaving the ability to produce a protein of the present invention, but ispreferably a microorganism belonging to the genus Bacillus, morepreferably a microorganism belonging to the genus Bacillus and havingthe bacilysin-synthesizing activity, further preferably a microorganismbelonging to a species selected from the group consisting of Bacillussubtilis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacilluslicheniformis, Bacillus megaterium and Bacillus pumilus, and mostpreferably a strain selected from the group consisting of Bacillussubtilis ATCC 15245, Bacillus subtilis ATCC 6633, Bacillus subtilis IAM1213, Bacillus subtilis IAM 1107, Bacillus subtilis IAM 1214, Bacillussubtilis ATCC 9466, Bacillus subtilis IAM 1033, Bacillus subtilis ATCC21555, Bacillus amyloliquefaciens IFO 3022 and Bacillus pumilus NRRLB-12025.

The treated matters of the culture include a concentrated culture, adried culture, cells obtained by centrifuging the culture, productsobtained by treating the cells by various means such as drying,freeze-drying, treatment with a surfactant, ultrasonication, mechanicalfriction, treatment with a solvent, enzymatic treatment, proteinfractionation and immobilization, an enzyme preparation obtained byextracting the cells, etc.

In the above process, the kinds of amino acids used as substrates, theirconcentrations, the time of their addition, and the dipeptides producedare similar to those in the enzymatic process described in the above(iii) (1).

In the process using a culture of a microorganism or a treated matter ofthe culture as an enzyme source, the culture of the transformant ormicroorganism used as an enzyme source can also be used as the aqueousmedium in addition to the aqueous media used in the enzymatic processdescribed in the above (iii) (1).

Further, in the above process, ATP or compounds which can be metabolizedby the transformant or microorganism to produce ATP, for example, sugarssuch as glucose, alcohols such as ethanol, and organic acids such asacetic acid may be added, as ATP source, to the aqueous medium accordingto need.

If necessary, a surfactant or an organic solvent may further be added tothe aqueous medium. Any surfactant that promotes the formation of adipeptide can be used. Suitable surfactants include nonionic surfactantssuch as polyoxyethylene octadecylamine (e.g., Nymeen S-215, NOFCorporation), cationic surfactants such as cetyltrimethylammoniumbromide and alkyldimethylbenzylammonium chloride (e.g., Cation F2-40E,NOF Corporation), anionic surfactants such as lauroyl sarcosinate, andtertiary amines such as alkyldimethylamine (e.g., Tertiary Amine FB, NOFCorporation), which may be used alone or in combination. The surfactantis usually used at a concentration of 0.1 to 50 g/l. As the organicsolvent, xylene, toluene, aliphatic alcohols, acetone, ethyl acetate,etc. may be used usually at a concentration of 0.1 to 50 ml/l.

When the culture or a treated matter of the culture is used as theenzyme source, the amount of the enzyme source to be added variesaccording to its specific activity, etc., but is, for example, 5 to 1000mg (wet cell weight), preferably 10 to 400 mg per mg of amino acid usedas a substrate.

The dipeptide-forming reaction is carried out in the aqueous medium atpH 5 to 11, preferably pH 6 to 10, at 20 to 65° C., preferably 25 to 55°C., more preferably 30 to 45° C., for 1 minute to 150 hours, preferably3 minutes to 120 hours, preferably 30 minutes to 100 hours.

In the processes described in the above (iii) (1) and (2), recovery ofthe dipeptide formed and accumulated in the aqueous medium can becarried out by ordinary methods using active carbon, ion-exchangeresins, etc. or by means such as extraction with an organic solvent,crystallization, thin layer chromatography and high performance liquidchromatography.

Certain embodiments of the present invention are illustrated in thefollowing examples. These examples are not to be construed as limitingthe scope of the invention.

EXAMPLE 1 Search for a Protein Having the Dipeptide-SynthesizingActivity utilizing a Database

By using, as a query, the amino acid sequence of D-Ala-D-Ala ligase genederived from Bacillus subtilis 168 [Nature, 390, 249-256 (1997)], asearch for a gene encoding a protein showing similarity which is presentin the genomic DNA sequences of Bacillus subtilis 168 was carried outusing the similarity search function of Subtilist(http://genolist.pasteur.fr/SubtiList/) which is a database of thegenomic DNA of Bacillus subtilis 168.

From the sequences obtained as a result of the search, genes encodingthe amino acid sequences shown in SEQ ID NOS: 33, 34 and 35 which areD-Ala-D-Ala ligase motifs [Biochemistry, 30, 1673 (1991)] and encodingproteins whose function had already been clarified were excluded. Of theremaining sequences, the sequence showing the highest similarity (29.1%)to the D-Ala-D-Ala ligase motif was selected as a gene of unknownfunction ywfE.

The nucleotide sequence of ywfE is shown in SEQ ID NO: 9, and the aminoacid sequence of the protein encoded by the nucleotide sequence is shownin SEQ ID NO: 1.

EXAMPLE 2 Construction of a Strain Expressing ywfE Gene

On the basis of the information on the nucleotide sequence obtained inExample 1, a ywfE gene fragment of Bacillus subtilis was obtained in thefollowing manner.

That is, Bacillus subtilis 168 (ATCC 23857) was inoculated into LBmedium [10 g/l Bacto-tryptone (Difco), 5 g/l yeast extract (Difco) and 5g/l sodium chloride] and subjected to static culture overnight at 30° C.After the culturing, the chromosomal DNA of the microorganism wasisolated and purified according to the method using saturated phenoldescribed in Current Protocols in Molecular Biology.

By using a DNA synthesizer (Model 8905, PerSeptive Biosystems, Inc.),DNAs having the nucleotide sequences shown in SEQ ID NOS: 19 to 22(hereinafter referred to as primer A, primer B, primer C and primer D,respectively) were synthesized. Primer A has a sequence wherein anucleotide sequence containing the XhoI recognition sequence is added tothe 5′ end of a region of the Bacillus subtilis chromosomal DNAcontaining the initiation codon of ywfE. Primer B has a sequence whereina nucleotide sequence containing the BamHI recognition sequence is addedto the 5′ end of a nucleotide sequence complementary to a sequencecontaining the termination codon of ywfE. Primer C has a sequencewherein a nucleotide sequence containing the EcoRI recognition sequenceis added to the 5′ end of the nucleotide sequence of trp promoter regionof expression vector pTrS30 containing trp promoter [prepared fromEscherichia coli JM109/pTrS30 (FERM BP-5407)]. Primer D has a sequencewherein a nucleotide sequence containing the XhoI recognition sequenceis added to the 5′ end of a sequence complementary to the sequence oftrp promoter region of expression vector pTrS30 containing trp promoter.

A ywfE gene fragment was amplified by PCR using the above primer A andprimer B, and as a template, the chromosomal DNA of Bacillus subtilis. Atrp promoter region fragment was amplified by PCR using primer C andprimer D, and as a template, pTrS30. PCR was carried out by 30 cycles,one cycle consisting of reaction at 94° C. for one minute, reaction at55° C. for 2 minutes and reaction at 72° C. for 3 minutes, using 40 μlof a reaction mixture comprising 0.1 μg of the chromosomal DNA or 10 ngof pTrS30 as a template, 0.5 μmol/l each of the primers, 2.5 units ofPfu DNA polymerase (Stratagene), 4 μl of buffer for Pfu DNA polymerase(10×) (Stratagene) and 200 μmol/l each of dNTPs (dATP, dGTP, dCTP anddTTP).

One-tenth of each of the resulting reaction mixtures was subjected toagarose gel electrophoresis to confirm that a ca. 1.4 kb DNA fragmentcorresponding to the ywfE gene fragment and a ca. 0.3 kb DNA fragmentcorresponding to the trp promoter region fragment were respectivelyamplified in the PCR using primer A and primer B and the PCR usingprimer C and primer D. Then, the remaining reaction mixture was mixedwith an equal amount of phenol/chloroform (1 vol/1 vol) saturated withTE [10 mmol/l Tris-HCl (pH 8.0), 1 mmol/l EDTA]. The resulting solutionwas centrifuged, and the obtained upper layer was mixed with a two-foldvolume of cold ethanol and allowed to stand at −80° C. for 30 minutes.The resulting solution was centrifuged to precipitate DNA, and theobtained DNA was dissolved in 20 μl of TE.

The thus obtained solutions (5 μl each) were respectively subjected toreaction to cleave the DNA amplified using primer A and primer B withrestriction enzymes XhoI and BamHI and to reaction to cleave the DNAamplified using primer C and primer D with restriction enzymes EcoRI andXhoI. DNA fragments were separated by agarose gel electrophoresis, and a1.4 kb fragment containing ywfE and a 0.3 kb fragment containing trppromoter region were respectively recovered using GENECLEAN II Kit (BIO101).

pTrs30 [a trp promoter-containing expression vector prepared fromEscherichia coli JM109/pTrS30 (FERM BP-5407), 0.2 μg] was cleaved withrestriction enzymes EcoRI and BamHI. DNA fragments were separated byagarose gel electrophoresis and a 4.5 kb DNA fragment was recovered inthe same manner as above.

The 1.4 kb fragment containing ywfE, the 0.3 kb fragment containing trppromoter region and the 4.5 kb DNA fragment obtained above weresubjected to ligation reaction using a ligation kit (Takara Shuzo Co.,Ltd.) at 16° C. for 16 hours.

Escherichia coli NM522 (Stratagene) was transformed using the reactionmixture according to the method using calcium ion [Proc. Natl. Acad.Sci. USA, 69, 2110 (1972)], spread on LB agar medium containing 50 μg/mlampicillin, and cultured overnight at 30° C.

A plasmid was extracted from a colony of the transformant that grew onthe medium according to a known method and the structure of the plasmidwas analyzed using restriction enzymes, whereby it was confirmed thatexpression vector pPE43 containing ywfE ligated downstream of the trppromoter was obtained (FIG. 1).

EXAMPLE 3 Production of a Dipeptide

Escherichia coli NM522 carrying pPE43 (Escherichia coli NM522/pPE43)obtained in Example 2 was inoculated into 8 ml of LB medium containing50 μg/ml ampicillin in a large test tube, and cultured at 28° C. for 17hours. The 10 resulting culture was centrifuged to obtain wet cells.

A reaction mixture (0.1 ml) comprising 60 mg/ml (final concentration)wet cells, 120 mmol/l potassium phosphate buffer (pH 7.4), 60 mmol/lmagnesium chloride, 60 mmol/l ATP, 30 mmol/l L-Ala, 30 mmol/l L-Gln and0.4% Nymeen S-215 was prepared, and reaction was carried out at 37° C.for 3 minutes.

After the completion of reaction, the reaction product was derivatizedby the dinitrophenol method and then analyzed by HPLC. The HPLC analysiswas carried out using, as a separation column, Lichrosorb-RP-18 column(Kanto Kagaku) and, as an eluent, 1% (v/v) phosphoric acid and 25% (v/v)acetonitrile at a flow rate of 0.7 ml/min. As a result, it was confirmedthat 120 mg/l L-alanyl-L-glutamine (L-Ala-L-Gln) was formed andaccumulated in the reaction mixture.

Formation of L-Ala-L-Gln was not observed when the reaction was carriedout using cells of Escherichia coli NM522/pTrS31, which is a controlstrain carrying only a vector.

EXAMPLE 4 Purification of C-Terminal His-Tagged Recombinant DipeptideSynthetase

By using the above DNA synthesizer, DNAs having the nucleotide sequencesshown in SEQ ID NOS: 23 and 24 (hereinafter referred to as primer E andprimer F, respectively) were synthesized. Primer E has a nucleotidesequence containing a region wherein the initiation codon of ywfE (atg)is substituted by the NcoI recognition sequence (ccatgg). Primer F has anucleotide sequence containing a region wherein the termination codon ofywfE is substituted by the BamHI recognition sequence (ggatcc).

PCR was carried out using the chromosomal DNA of Bacillus subtilis 168(ATCC 23857) as a template and the above primer E and primer F as a setof primers. That is, PCR was carried out by 30 cycles, one cycleconsisting of reaction at 94° C. for one minute, reaction at 55° C. for2 minutes and reaction at 72° C. for 3 minutes, using 40 μl of areaction mixture comprising 0.1 μg of the chromosomal DNA, 0.5 μmol/leach of the primers, 2.5 units of Pfu DNA polymerase, 4 μl of buffer forPfu DNA polymerase (10×) and 200 μmol/l each of dNTPs.

One-tenth of the resulting reaction mixture was subjected to agarose gelelectrophoresis to confirm that a ca. 1.4 kb fragment corresponding tothe ywfE fragment was amplified. Then, the remaining reaction mixturewas mixed with an equal amount of phenol/chloroform saturated with TE.The resulting solution was centrifuged, and the obtained upper layer wasmixed with a two-fold volume of cold ethanol and allowed to stand at−80° C. for 30 minutes. The resulting solution was centrifuged, and theobtained DNA precipitate was dissolved in 20 μl of TE.

The thus obtained solution (5 μl) was subjected to reaction to cleavethe amplified DNA with restriction enzymes NcoI and BamHI. DNA fragmentswere separated by agarose gel electrophoresis, and a 1.4 kb DNA fragmentcontaining ywfE was recovered using GENECLEAN II Kit.

C-Terminal His-tagged recombinant expression vector pQE60 (Qiagen, Inc.)(0.2 μg) was cleaved with restriction enzymes NcoI and BamHI. DNAfragments were separated by agarose gel electrophoresis, and a 3.4 kbDNA fragment was recovered in the same manner as above.

The 1.4 kb DNA fragment containing ywfE and the 3.4 kb DNA fragmentobtained above were subjected to ligation reaction using a ligation kitat 16° C. for 16 hours.

Escherichia coli NM522 was transformed using the ligation reactionmixture according to the method using calcium ion, spread on LB agarmedium containing 50 μg/ml ampicillin, and cultured overnight at 30° C.

A plasmid was extracted from a colony of the transformant that grew onthe medium according to a known method and the structure of the plasmidwas analyzed using restriction enzymes, whereby it was confirmed thatpQE60ywfE, which is a C-terminal His-tagged ywfE expression vector, wasobtained (FIG. 2).

Escherichia coli NM522 carrying pQE60ywfE (Escherichia coliNM522/pQE60ywfE) was inoculated into 8 ml of LB medium containing 50μg/ml ampicillin in a large test tube, and cultured at 28° C. for 17hours. The resulting culture was inoculated into 50 ml of LB mediumcontaining 50 μg/ml ampicillin in a 250-ml Erlenmeyer flask, andcultured at 30° C. for 3 hours. Then,isopropyl-β-D-thiogalactopyranoside (IPTG) was added to give a finalconcentration of 1 mmol/l, followed by further culturing at 30° C. for 4hours. The resulting culture was centrifuged to obtain wet cells, and aHis-tagged recombinant enzyme was purified from the wet cells usingHisTrap (His-tagged protein purification kit, Amersham PharmaciaBiotech) according to the instructions attached thereto.

EXAMPLE 5 Production of Dipeptides Using the His-Tagged RecombinantEnzyme (1)

(i) A reaction mixture (0.1 ml) comprising 0.04 mg of the purifiedHis-tagged recombinant enzyme obtained in Example 4, 100 mmol/l Tris-HCl(pH 8.0), 60 mmol/l magnesium chloride, 60 mmol/l ATP, 30mmol/l L-Alaand 30 mmol/l L-Gin was prepared, and reaction was carried out at 37° C.for 16 hours.

After the completion of reaction, the reaction product was analyzed inthe same manner as in Example 3 above, whereby it was confirmed that 3.7g/l L-Ala-L-Gln and 0.3 g/l L-alanyl-L-alanine (L-Ala-L-Ala) were formedand accumulated in the reaction mixture.

(ii) Reactions were carried out under the same conditions as in theabove (i) using reaction mixtures having the same composition as that ofthe reaction mixture of the above (i) except that 0.01 mg of the enzymewas used and L-Phe, L-Met, L-Leu and L-Val, respectively, were used inplace of L-Gln.

After the completion of reactions, the reaction products were analyzedin the same manner as in Example 3 above, whereby it was confirmed thatthe following dipeptides were formed and accumulated in the respectivereaction mixtures: 7.0 g/l L-alanyl-L-phenylalanine (L-Ala-L-Phe) alone;7.0 g/l L-alanyl-L-methionine (L-Ala-L-Met) and 0.03 g/l L-Ala-L-Ala;5.0 g/l L-alanyl-L-leucine (L-Ala-L-Leu) and 0.2 g/l L-Ala-L-Ala; and1.6 g/l L-alanyl-L-valine (L-Ala-L-Val) and 0.3 g/l L-Ala-L-Ala.

(iii) Reactions were carried out under the same conditions as in theabove (i) using reaction mixtures having the same composition as that ofthe reaction mixture of the above (i) except that 0.01 mg of the enzymewas used, Gly was used in place of L-Ala, and L-Phe and L-Met,respectively, were used in place of L-Gln.

After the completion of reactions, the reaction products were analyzedin the same manner as in Example 3 above, whereby it was confirmed that5.2 g/l glycyl-L-phenylalanine (Gly-L-Phe) and 1.1 g/lglycyl-L-methionine (Gly-L-Met) were formed and accumulated in therespective reaction mixtures.

When ATP was excluded from the compositions of the above reactionmixtures, no dipeptide was formed.

The above results revealed that the ywfE gene product has the activityto produce, in the presence of ATP, the following dipeptides:L-Ala-L-Gln plus L-Ala-L-Ala, L-Ala-L-Phe, L-Ala-L-Met plus L-Ala-L-Ala,L-Ala-L-Leu plus L-Ala-L-Ala, or L-Ala-L-Val plus L-Ala-L-Ala from L-Alaplus L-Gln, L-Phe, L-Met, L-Leu or L-Val; and Gly-L-Phe or Gly-L-Metfrom Gly plus L-Phe or L-Met.

EXAMPLE 6 Production of Dipeptides Using the His-Tagged RecombinantEnzyme (2)

A reaction mixture (0.1 ml) comprising 0.04 mg of the purifiedHis-tagged recombinant enzyme obtained in Example 4, 100 mmol/l Tris-HCl(pH 8.0), 60 mmol/l magnesium chloride and 60 mmol/l ATP was prepared.To this mixture were respectively added combinations of L-amino acids,Gly or β-Ala shown in the first row of Table 1 and L-amino acids, Gly orβ-Ala shown in the leftmost column of Table 1 to give a concentration of30 mmol/l each, and the resulting mixtures were subjected to reaction at37° C. for 16 hours. After the completion of reactions, the reactionproducts were analyzed by HPLC, whereby it was confirmed that thedipeptides shown in Table 1 were formed.

TABLE 1

The dipeptides formed by the reaction using, as substrates, two (or one)kinds of L-amino acids, Gly or β-Ala shown in the first row and theleftmost column of Table 1 are shown in the respective cell of thetable. In the table, ◯ means that a dipeptide was formed though itssequence was unidentified; X means that formation of a dipeptide was notconfirmed; and a blank means that reaction was not carried out.

EXAMPLE 7 Production of a Dipeptide using the Strain Expressing theHis-Tagged Recombinant Enzyme

Escherichia coli NM522/pQE60ywfE obtained in Example 4 was inoculatedinto 8 ml of LB medium containing 50 μg/ml ampicillin in a large testtube, and cultured at 28° C. for 17 hours. The resulting culture wasinoculated into 50 ml of LB medium containing 50 μg/ml ampicillin in a250-ml Erlenmeyer flask, and cultured at 30° C. for 3 hours. Then,isopropyl-β-D-thiogalactopyranoside (IPTG) was added to give a finalconcentration of 1 mmol/l, followed by further culturing at 30° C. for 4hours. The resulting culture was centrifuged to obtain wet cells.

A reaction mixture (20 ml, pH 7.2) comprising 200 g/l wet cells, 50 g/lglucose, 5 g/l phytic acid (diluted to neutrality with 33% conc. sodiumhydroxide solution), 15 g/l potassium dihydrogenphosphate, 5 g/lmagnesium sulfate heptahydrate, 4 g/l Nymeen S-215, 10 ml/l xylene, 200mmol/l L-Ala and 200 mmol/l L-Gln was put in a 50-ml beaker, andreaction was carried out at 32° C. at 900 rpm for 2 hours. During thereaction, the pH of the reaction mixture was maintained at 7.2 by using2 mol/l potassium hydroxide.

The reaction product was analyzed by the same method as in Example 3,whereby it was confirmed that 25 mg/l L-Ala-L-Gln was accumulated.

EXAMPLE 8 Cloning of Genes Corresponding to the ywfE Gene from VariousMicroorganisms of the Genus Bacillus and Analysis Thereof

On the basis of the nucleotide sequence shown in SEQ ID NO: 9, genescorresponding to the ywfE gene which exist in Bacillus subtilis ATCC15245, ATCC 6633, IAM 1213, IAM 1107, IAM 1214, ATCC 9466, IAM 1033 andATCC 21555, Bacillus amyloliquefaciens IFO 3022 and Bacillus pumilusNRRL B-12025 were obtained in the following manner.

That is, Bacillus subtilis ATCC 15245, ATCC 6633, IAM 1213, IAM 1107,IAM 1214, ATCC 9466, IAM 1033 and ATCC 21555, Bacillus amyloliquefaciensIFO 3022 and Bacillus pumilus NRRL B-12025 were respectively inoculatedinto LB medium and subjected to static culture overnight at 30° C. Afterthe culturing, the chromosomal DNAs of the respective microorganismswere isolated and purified according to the method using saturatedphenol described in Current Protocols in Molecular Biology.

By using a DNA synthesizer (Model 8905, PerSeptive Biosystems, Inc.),DNAs having the nucleotide sequences shown in SEQ ID NOS: 25 and 26(hereinafter referred to as primer G and primer H, respectively) weresynthesized. Primer G has a sequence containing a region upstream of theinitiation codon of ywfE of the chromosomal DNA of Bacillus subtilis168, and primer H has a sequence complementary to a sequence containinga region downstream of the termination codon of ywfE.

PCR was carried out using each of the chromosomal DNAs of Bacillussubtilis ATCC 15245, ATCC 6633, IAM 1213, IAM 1107, IAM 1214, ATCC 9466,IAM 1033 and ATCC 21555 and Bacillus amyloliquefaciens IFO 3022 as atemplate and the above primer G and primer H as a set of primers. Thatis, PCR was carried out by 30 cycles, one cycle consisting of reactionat 94° C. for one minute, reaction at 55° C. for 2 minutes and reactionat 72° C. for 3 minutes, using 40 μl of a reaction mixture comprising0.1 μg of the chromosomal DNA, 0.5 μmol/l each of the primers, 2.5 unitsof Pfu DNA polymerase, 4 μl of buffer for Pfu DNA polymerase (10×) and200 μmol/l each of dNTPs.

One-tenth of each of the resulting reaction mixtures was subjected toagarose gel electrophoresis to confirm that a ca. 1.4 kb fragmentcorresponding to the ywfE fragment was amplified. Then, the remainingreaction mixture was mixed with an equal amount of phenol/chloroformsaturated with TE. The resulting solution was centrifuged, and theobtained upper layer was mixed with a two-fold volume of cold ethanoland allowed to stand at −80° C. for 30 minutes. The resulting solutionwas centrifuged, and the obtained DNA precipitate was dissolved in 20 μlof TE.

Each of the thus obtained 1.4 kb DNA fragments derived from thechromosomal DNAs of the respective strains and pCR-blunt (InvitrogenCorp.) were subjected to ligation reaction using a ligation kit at 16°C. for 16 hours.

Escherichia coli NM522 was transformed using each ligation reactionmixture according to the method using calcium ion, spread on LB agarmedium containing 50 μg/ml ampicillin, and cultured overnight at 30° C.

A plasmid was extracted from a colony of each transformant that grew onthe medium according to a known method and the structure of each plasmidwas analyzed using restriction enzymes. As a result, it was confirmedthat the following plasmids containing a gene corresponding to the ywfEgene were obtained: pYWFE1 (derived from ATCC 15245(SEQ ID NO: 36)),pYWFE2 (derived from ATCC 6633(SEQ ID NO: 10)), pYWFE3 (derived from IAM1213(SEQ ID NO: 11)), pYWFE4 (derived from IAM 1107(SEQ ID NO: 12)),pYWFE5 (derived from IAM 1214(SEQ ID NO: 13)), pYWFE6 (derived from ATCC9466), pYWFE7 (derived from IAM 1033(SEQ ID NO: 36)), pYWFE8 (derivedfrom ATCC 21555(SEQ ID NO: 14)) and pYWFE9 (derived from IFO 3022(SEQ IDNO: 15)).

On the other hand, a gene corresponding to ywfE derived from Bacilluspumilus NRRL B-12025(SEQ ID NO: 16) was obtained in the followingmanner.

PCR was carried out using the chromosomal DNA of the NRRL B-12025 strainprepared above as a template and DNAs respectively consisting of thenucleotide sequences shown in SEQ ID NOS: 27 and 28 as a set of primers.That is, PCR was carried out by 30 cycles, one cycle consisting ofreaction at 98° C. for 5 seconds, reaction at 55° C. for 30 seconds andreaction at 72° C. for one minute, using 50 μl of a reaction mixturecomprising 0.1 μg of the chromosomal DNA, 0.5 μmol/l each of theprimers, 2.5 units of Z-taq polymerase (Takara Shuzo Co., Ltd.), 5 μl ofbuffer for Z-taq polymerase (10×) (Takara Shuzo Co., Ltd.) and 200μmol/l each of dNTPs.

One-tenth of the resulting reaction mixture was subjected to agarose gelelectrophoresis to confirm that a ca. 0.8 kb fragment was amplified.Then, the remaining reaction mixture was mixed with an equal amount ofphenol/chloroform saturated with TE. The resulting mixture wascentrifuged, and the obtained upper layer was mixed with a two-foldvolume of cold ethanol and allowed to stand at −80° C. for 30 minutes.The resulting solution was centrifuged, and the obtained DNA precipitatewas dissolved in 20 μl of TE.

The thus obtained 0.8 kb fragment derived from the chromosomal DNA andpGEM T-easy (Promega Corp.) were subjected to ligation reaction using aligation kit at 16° C. for 16 hours.

Escherichia coli DH5α was transformed using the reaction mixtureaccording to the method using calcium ion, spread on LB agar mediumcontaining 50 μg/ml ampicillin, and cultured overnight at 30° C.

A plasmid was extracted from the transformant obtained above and thenucleotide sequence of the ca. 0.8 kb DNA insert was determined, wherebya sequence from nucleotides 358 to 1160 in the nucleotide sequence shownin SEQ ID NO: 16 was confirmed.

The above plasmid was cleaved with EcoRI and then subjected to agarosegel electrophoresis to separate a DNA fragment. The DNA fragment waspurified using GENECLEAN II Kit, and about 0.5 μg of the purified DNAfragment was DIG-labeled using DIG-High Prime DNA Labeling & DetectionStarter Kit I (Roche Diagnostics Corp.) according to the instructionsattached thereto.

Southern analysis of the chromosomal DNA of the NRRL B-12025 strain wascarried out using the DIG-labeled DNA obtained above.

The chromosomal DNA of the NRRL B-12025 strain was completely digestedwith BamHI, EcoRI, HindIII, KpnI, PstI, SacI, SalI and SphI,respectively, and subjected to agarose gel electrophoresis to separateDNA fragments, followed by transfer to nylon membrane plus charge (RocheDiagnostics Corp.) according to an ordinary method.

After the DNA fragments were fixed on the nylon membrane by UVirradiation, Southern hybridization was carried out using the aboveprobe DNA and the nylon membrane. The hybridization was carried out bybringing the nylon membrane into contact with the probe DNA at 65° C.for 16 hours, washing the nylon membrane twice with a solutionconsisting of 0.1% SDS and 2×SSC at room temperature for 5 minutes, andfurther washing the membrane twice with a solution consisting of 0.1%SDS and 0.5×SSC at 65° C. for 15 minutes. The other operations andconditions and detection of the hybridized DNA were carried outaccording to the instructions attached to the above-mentioned DIG-HighPrime DNA Labeling & Detection Starter Kit I.

As a result, color development was observed at around 3.5 kbp of thefragments completely digested with HindIII and PstI.

Subsequently, the chromosomal DNA of the NRRL B-12025 strain wascompletely digested with HindIII and PstI, respectively, and subjectedto agarose gel electrophoresis to separate DNA fragments. From therespective restriction enzyme-digested DNAs, 3-4 kbp fragments werepurified using GENECLEAN II Kit, followed by autocyclization using aligation kit.

On the basis of the nucleotide sequence of the 0.8 kb DNA fragmentdetermined above, the nucleotide sequences shown in SEQ ID NOS: 29 and30 were designed and synthesized, and they were used in PCR, as primers,using the cyclized DNA obtained above as a template. PCR was carried outby 30 cycles, one cycle consisting of reaction at 98° C. for 5 seconds,reaction at 55° C. for 30 seconds and reaction at 72° C. for 3 minutesand 30 seconds, using 50 μl of a reaction mixture comprising 10 ng ofthe cyclized DNA, 0.5 μmol/l each of the primers, 2.5 units of pyrobestpolymerase (Takara Shuzo Co., Ltd.), 5 μl of buffer for pyrobestpolymerase (10×) (Takara Shuzo Co., Ltd.) and 200 μmol/l each of dNTPs.

One-tenth of the resulting reaction mixture was subjected to agarose gelelectrophoresis to confirm that a ca. 3.0 kb fragment was amplified.Then, the remaining reaction mixture was mixed with an equal amount ofphenol/chloroform saturated with TE. The resulting mixture wascentrifuged, and the obtained upper layer was mixed with a two-foldvolume of cold ethanol and allowed to stand at −80° C. for 30 minutes.The resulting solution was centrifuged, and the obtained DNA precipitatewas dissolved in 20 μl of TE.

The thus obtained DNA fragment and Zero Blunt PCR Cloning Kit(Invitrogen Corp.) were subjected to ligation reaction using a ligationkit.

Escherichia coli NM522 was transformed using the reaction mixtureaccording to the method using calcium ion, spread on LB agar mediumcontaining 50 μg/ml ampicillin, and cultured overnight at 30° C.

A plasmid was extracted from a colony of the transformant that grew onthe medium according to a known method and the structure of the plasmidwas analyzed using restriction enzymes. As a result, it was confirmedthat plasmid pYWFE10 (derived from NRRL B-12025) containing a genecorresponding to the ywfE gene was obtained.

The nucleotide sequences of the genes corresponding to the ywfE genewhich are respectively contained in the plasmids pYWFE1 to pYWFE10obtained above were determined using 373A DNA Sequencer.

The amino acid sequences of the proteins encoded by the genesrespectively contained in pYWFE1, pYWFE6 and pYWFE7 were identical withthe amino acid sequence of the protein encoded by the ywfE gene, whereasthose of the proteins encoded by the genes respectively contained inpYWFE2, pYWFE3, pYWFE4, pYWFE5, pYWFE8, pYWFE9 and pYWFE10 weredifferent from the amino acid sequence of the protein encoded by theywfE gene.

The amino acid sequences of the proteins encoded by the genescorresponding to ywfE which are contained in pYWFE2, pYWFE3, pYWFE4,pYWFE5, pYWFE8, pYWFE9, pYWFE10, and pYWFE1 and pYWFE7 are shown in SEQID NOS: 2 to 8 and 1, respectively, and the nucleotide sequences ofthese genes are shown in SEQ ID NOS: 10 to 16 and 36, respectively.

EXAMPLE 9 Purification of C-Terminal His-Tagged Recombinant DipeptideSynthetase

PCR was carried out using each of the chromosomal DNAS of Bacillussubtilis ATCC 15245, ATCC 6633, IAM 1213, IAM 1107, IAM 1214, ATCC 9466,IAM 1033 and ATCC 21555 and Bacillus amyloliquefaciens IFO 3022 as atemplate and primer A and primer B described in Example 2 as a set ofprimers. That is, PCR was carried out by 30 cycles, one cycle consistingof reaction at 94° C. for one minute, reaction at 55° C. for 2 minutesand reaction at 72° C. for 3 minutes, using 40 μl of a reaction mixturecomprising 0.1 μg of the chromosomal DNA, 0.5 μmol/l each of theprimers, 2.5 units of Pfu DNA polymerase, 4 μl of buffer for Pfu DNApolymerase (10×) and 200 μmol/l each of dNTPs.

When the chromosomal DNA of Bacillus pumilus NRRL B-12025 was used as atemplate, PCR was carried out using DNAs respectively having thenucleotide sequences shown in SEQ ID NOS: 31 and 32 as a set of primersunder the same conditions as above.

One-tenth of each of the resulting reaction mixtures was subjected toagarose gel electrophoresis to confirm that a ca. 1.4 kb DNA fragmentcorresponding to the ywfE fragment was amplified. Then, the remainingreaction mixture was mixed with an equal amount of phenol/chloroformsaturated with TE. The resulting mixture was centrifuged, and theobtained upper layer was mixed with a two-fold volume of cold ethanoland allowed to stand at −80° C. for 30 minutes. The resulting solutionwas centrifuged, and the obtained DNA precipitate was dissolved in 20 μlof TE.

Each of the thus obtained solutions (5 μl) was subjected to reaction tocleave the amplified DNA with restriction enzymes NcoI and BamHI. DNAfragments were separated by agarose gel electrophoresis, and a 1.4 kbDNA fragment containing a gene corresponding to ywfE was recovered usingGENECLEAN II Kit.

Subsequently, 0.2 μg of the C-terminal His-tagged recombinant expressionvector pQE60 was cleaved with restriction enzymes NcoI and BamHI. DNAfragments were separated by agarose gel electrophoresis, and a 3.4 kbDNA fragment was recovered in the same manner as above.

Each of the 1.4 kb DNA fragments containing a gene corresponding to ywfEof Bacillus subtilis 168 and the 3.4 kb DNA fragment obtained above weresubjected to ligation reaction using a ligation kit at 16° C. for 16hours.

Escherichia coli NM522 was transformed using each ligation reactionmixture according to the method using calcium ion, spread on LB agarmedium containing 50 μg/ml ampicillin, and cultured overnight at 30° C.

A plasmid was extracted from a colony of each transformant that grew onthe medium according to a known method and the structure of each plasmidwas analyzed using restriction enzymes. As a result, it was confirmedthat the following C-terminal His-tagged gene expression vectors wereobtained: pQE60ywfE1 (a vector containing the gene derived from ATCC15245), pQE60ywfE2 (a vector containing the gene derived from ATCC6633), pQE60ywfE3 (a vector containing the gene derived from IAM 1213),pQE60ywfE4 (a vector containing the gene derived from IAM 1107),pQE60ywfE5 (a vector containing the gene derived from IAM 1214),pQE60ywfE6 (a vector containing the gene derived from ATCC 9466),pQE60ywfE7 (a vector containing the gene derived from IAM 1033),pQE60ywfE8 (a vector containing the gene derived from ATCC 21555),pQE60ywfE9 (a vector containing the gene derived from IFO 3022) andpQE60ywfE10 (a vector containing the gene derived from NRRL B-12025).

Escherichia coli NM522/pQE60ywfE1 to NM522/pQE60ywfE10 strains obtainedabove were respectively inoculated into 8 ml of LB medium containing 50μg/ml ampicillin in a large test tube, and cultured at 28° C. for 17hours. Each of the resulting cultures was inoculated into 50 ml of LBmedium containing 50 μg/ml ampicillin in a 250-ml Erlenmeyer flask, andcultured at 30° C. for 3 hours. Then,isopropyl-β-D-thiogalactopyranoside was added to give a finalconcentration of 1 mmol/l, followed by further culturing at 30° C. for 4hours. The resulting culture was centrifuged to obtain wet cells, andHis-tagged recombinant enzymes were purified from the respective wetcells using HisTrap according to the instructions attached thereto.

EXAMPLE 10 Production of Dipeptides Using Purified Enzymes

Reaction mixtures (0.1 ml each) comprising 0.04 mg of the respectiverecombinant enzymes obtained in Example 9, 100 mmol/l Tris-HCl (pH 8.0),60 mmol/l magnesium chloride, 60 mmol/l ATP, 30 mmol/l L-Ala and 30mmol/l L-Gln were prepared, and reactions were carried out at 37° C. for16 hours.

After the completion of reactions, the reaction mixtures were analyzedby the method described in Example 3, whereby it was confirmed that 3.0to 3.5 g/l L-Ala-L-Gln and 0.25 to 0.3 g/l L-Ala-L-Ala were formed andaccumulated.

When ATP was excluded from the compositions of the above reactionmixtures, L-Ala-L-Gln or L-Ala-L-Ala was not formed at all.

The above results revealed that all of the products of the genesobtained in Example 8 have the activity to produce L-Ala-L-Gln andL-Ala-L-Ala from L-Ala and L-Gln in the presence of ATP.

The entire content of each reference, application, publication, patentand document cited herein is hereby incorporated herein in its entirelyby reference.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO: 19—Description of Artificial Sequence: Primer-   SEQ ID NO: 20—Description of Artificial Sequence: Primer-   SEQ ID NO: 21—Description of Artificial Sequence: Primer-   SEQ ID NO: 22—Description of Artificial Sequence: Primer-   SEQ ID NO: 23—Description of Artificial Sequence: Primer-   SEQ ID NO: 24—Description of Artificial Sequence: Primer-   SEQ ID NO: 25—Description of Artificial Sequence: Primer-   SEQ ID NO: 26—Description of Artificial Sequence: Primer-   SEQ ID NO: 27—Description of Artificial Sequence: Primer-   SEQ ID NO: 28—Description of Artificial Sequence: Primer-   SEQ ID NO: 29—Description of Artificial Sequence: Primer-   SEQ ID NO: 30—Description of Artificial Sequence: Primer-   SEQ ID NO: 31—Description of Artificial Sequence: Primer-   SEQ ID NO: 32—Description of Artificial Sequence: Primer-   SEQ ID NO: 33—Description of Artificial Sequence: Amino acid    sequence used in database search-   SEQ ID NO: 34—Description of Artificial Sequence: Amino acid    sequence used in database search-   SEQ ID NO: 35—Description of Artificial Sequence: Amino acid    sequence used in database search

1. A process for producing a dipeptide represented by formula (I):R-R2  (I) wherein R1 is an amino acid selected from the group consistingof L-alanine, L-methionine, L-serine, β-alanine, L-threonine, glycine,L-cysteine and L-α-aminobutyric acid, and R² is an amino acid selectedfrom the group consisting of L-alanine, L-glutamine, L-glutamic acid,glycine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine,L-tryptophan, L-methionine, L-serine, L-threonine, L-cysteine,L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-asparticacid, L-α-aminobutyric acid, βalanine, L-azaserine, L-theanine, L-4-hydroxyprol me, L-3-hydroxyproline, L-ornithine, L-citrulline andL-6-diazo-5-oxo-norleucine except L-alanyl-L-alanine, which comprises:contacting, in an aqueous medium, at least two amino acids which may bethe same or different, and a transformed microorganism comprising apolynucleotide encoding a protein selected from the group consisting ofthe following [1]to [3 ]: [1]a protein comprising the amino acidsequence of SF0 ID NO:1; [2]a protein comprising an amino acid sequencewherein 1 to 20 amino acid residues are deleted, substituted or added inthe amino acid sequence of SEQ ID NO: 1 and catalyzing the synthesis ofa dipeptide represented by formula (I); and [3]a protein comprising anamino acid sequence which shows 98% or more similarity determined byBLAST program to the amino acid sequence of SEQ ID NO: 1 and catalyzesthe synthesis of a dipeptide represented by formula (I); forming andaccumulating the dipeptide in the medium; and recovering the dipeptidefrom the medium.
 2. A process for producing a dipeptide represented byformula (I);R¹-R²  (I) wherein R¹ is an amino acid selected from the groupconsisting of L-alanine, L-methionine, L-serine, β-alanine, L-threonine,glycine, L-cysteine and L-α-aminobutyric acid, and R2 is an amino acidselected from the group consisting of L-alanine, L-glutamine, L-glutamicacid, glycine, L-valine, L-leucine, L-isoleucine, L-proline,L-phenylalanine, L-tryptophan, L-methionine, L-serine, L-threonine,L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine,L-aspartic acid, L-α-aminobutyric acid, β-alanine, L-azaserine,L-theanine, L-4-hydroxyproline, L-3-hydroxyproline, L-ornithine,L-citrulline and L-6-diazo-5 -oxo-norleucine except L-alanyl-L-alanine,which comprises: contacting an enzyme source and at least two aminoacids which may be the same or different in an aqueous medium, saidenzyme source being a culture of a microorganism transformed with anucleic acid sequence selected from the group consisting of (a) to (e),or a treated matter of the culture: (a) a nucleic acid sequence encodingthe protein comprising the amino acid sequence of SEQ ID NO:1; (b) anucleic acid comprising the nucleotide sequence of SEQ ID NO:9; (c) anucleic acid sequence encoding a protein comprising an amino acidsequence wherein 1 to 20 amino acid residues are deleted, substituted oradded in the amino acid sequence of SEQ ID NO: 1 and catalyzing thesynthesis of the dipeptide represented by formula (I); and (d) a nucleicacid sequence which hybridizes with a nucleic acid sequence comprisingthe complement of a nucleotide sequence of SEQ ID NOS: 9 in the presenceof 0.7 to 1.0 mol/l sodium chloride at 65° C., followed by washing in0.1 to 2-fold concentrated SSC solution at 65° C. and which nucleic acidsequence encodes a protein which catalyzes the synthesis of a dipeptiderepresented by formula (I); and (e) a protein comprising an amino acidsequence which shows 98% or more similarity to the amino acid sequenceof SEQ ID NO: 1 and catalyzes the synthesis of the dipeptide representedby formula (I); forming and accumulating the dipeptide in the medium;and recovering the dipeptide from the medium wherein the treated matterof the culture is selected from the group consisting of a concentratedculture, a dried culture, cells obtained by centrifuging the culture anda product obtained by subjecting the cells to drying, freeze-drying,treatment with a surfactant and treatment with a solvent, and thetreated matter of the culture has same enzymatic activity as theculture.
 3. The process according to claim 2, wherein the microorganismis transformed with DNA derived from the genus Bacillus.
 4. The processaccording to claim 3, wherein the microorganism belonging to the genusBacillus has the ability to produce bacilysin.
 5. The process accordingto claim 4, wherein the microorganism of the genus Bacillus is selectedfrom the group consisting of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillusmegaterium and Bacillus pumilus.
 6. The process according to claim 2,wherein the microorganism is transformed with a recombinant nucleic acidsequence comprising the nucleotide sequence of SEQ ID NO:
 9. 7. Theprocess according to claim 6, wherein the microorganism transformed witha nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO:9 belongs to the genus Escherichia.